Biological systems input-output response system and plant sentinels

ABSTRACT

A eukaryotic input circuit: computationally designed receptors, synthetic eukaryotic signal transduction pathways, and a synthetic signal sensitive promoter that allow highly specific transcriptional induction in response to an externally provided ligand is disclosed. The input circuit is able to specifically bind a targeted substance and transmit a signal to the nucleus where transcription of a gene is activated. An output circuit serves as a simple readout system of the substance detected by the input circuit. The readout circuit exemplified here is a degreening circuit which causes plants to turn white. Activation of the degreening circuit can be detected by eye, or remotely with a variety of machines (hand-held, aircraft or satellite based) and is also resettable. When linked the input circuit if operably linked to the output circuit, produces a functional plant detector.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 from U.S.Application Ser. No. 60/795,614 filed Apr. 26, 2006, which isincorporated herein in its entirety by reference.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with government support underN00014-03-1-0567 awarded by the United States Navy. Accordingly, theUnited States government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference a file named 1511-002Medford sequences.ST25.txt including SEQ ID NO:1 to SEQ ID NO:41,provided in a computer readable form and filed with the presentapplication. The sequence listing recorded on the CD-ROM is identical tothe written (on paper) sequence listing provided herein.

BACKGROUND OF THE INVENTION

The present invention relates to plant molecular biology, to signaltransduction from outside the plant to the nucleus and to systems forsensing a target substance of interest in the environment and inducinggene expression in response thereto, along with a novel plant biomarkeruseful for reporting the detection of biological and chemical agents andenvironmental pollutants based on the loss of green color in plants. Allpublications cited in this application are herein incorporated byreference.

There have been increasing threats from terrorists which present anurgent need for simple and robust detectors for harmful biological orchemical agents. Current detectors of biological and chemical agentsinvolve electronic and/or vacuum-like mechanisms to sample the air orthe environment. All current means to detect terrorist agents are costlyand require continuous maintenance. The high and continuous costsignificantly limits the ability to detect biological and chemicalweapons as well as environmental pollutants.

Each fall, plants display a dramatic loss in green color. The brightyellow, red, and orange colors are unmasked by the tremendous loss inthe green chlorophyll pigments. Consequently, the loss of green color inplants is a phenomenon that most people readily recognize. Thismetabolic degreening process is not unique to the foliage of deciduoustrees. A metabolic degreening is found in all plants includingevergreens and algae (Matile et al., 1999).

The green color in plants is due to a pigment known as chlorophyll. Mosthigher green plants contain two types of chlorophyll, chlorophyll a andchlorophyll b. Each molecule has a porphyrin-like ring attached to along hydrocarbon tail. Chlorophyll a and chlorophyll b differ only in aside group of ring II. In most plants, chlorophyll a is the dominantform with lesser amounts of chlorophyll b. The two forms of chlorophyllundergo a simple cyclic interconversion. Chlorophyll is synthesized inthe a form and can be converted to the b form through chlorophyll aoxygenase; chlorophyll b is converted back to chlorophyll a through theaction of chlorophyll b reductase (Malkin and Niyogi, 2000; Thomas etal., 2002).

The biosynthetic pathway for chlorophyll is very well known (Malkin andNiyogi, 2000). Chlorophyll biosynthesis begins with glutamic acid.Through nine biochemical steps glutamic acid is converted to a four-ringstructure, protoporphyrin IX. Magnesium chelatase adds magnesium to thering structure. In two additional steps, monovinyl protochlorophyllide ais formed. The enzyme protochlorophyllide oxidoreductase (POR) reducesthe monovinyl protochlorophyllide molecule to form chlorophyllide a.Importantly, the POR enzyme controls the rate-limiting step inchlorophyll biosynthesis. Chlorophyllide a has a light green color anddiffers from chlorophyll by lacking the long hydrocarbon tail. Thechlorophyllide molecule is converted to the darker green chlorophyllmolecule by the enzyme chlorophyll synthetase, which adds atwenty-carbon phytol tail. Like most biological molecules, steady statelevels of chlorophyll are maintained by a combination of biosynthesisand catabolism with the half-life of chlorophyll in a green plant beingapproximately 50 hours (Matile et al., 1999).

Like chlorophyll biosynthesis, the chlorophyll breakdown pathway is alsovery well characterized (Matile et al., 1999; Tsuchiya et al., 1999;Dangl et al., 2000). Chlorophyllase, one of the major enzymes involvedin the first step of chlorophyll breakdown, removes the hydrophobic,twenty carbon phytol tail from chlorophyll (Matile et al., 1999;Tsuchiya et al., 1999; Dangl et al., 2000; Benedetti and Arruda, 2002).Similar to the biosynthetic pathway, chlorophyll without the phytol tailbecomes the light green molecule, chlorophyllide. The lack of the phytoltail also changes solubility; chlorophyllide is soluble in aqueoussolutions whereas chlorophyll is soluble in organic solvents.

The chlorophyllide a molecule is converted to pheophorbide a by removalof the magnesium by the enzyme magnesium dechelatase (Matile et al.,1999; Dangl et al., 2000; Takamiya et al., 2000). A red-coloredcompound, red chlorophyll catabolite (RCC), forms next through theaction of the enzyme pheophorbide a oxygenase (Hortensteiner et al.,1998; Thomas et al., 2002). Next, the enzyme RCC reductase acts toproduce fluorescent chlorophyll catabolite (FCC). Subsequently, variousenzymes convert FCC to nonfluorescent chlorophyll catabolites.Nonfluorescent chlorophyll catabolite molecules accumulate in theplant's vacuole.

Importantly, the chlorophyll degradation pathway is not thought to bepart of the system involved in steady-state regulation of chlorophylllevels, because chlorophyll catabolites have never been found in greencells with steady chlorophyll levels (Matile et al., 1999). Thissuggests that induction of genes in the chlorophyll degradation pathwaywill lead to the rapid breakdown of chlorophyll. Indeed, transgenicplants with constitutive expression of the chlorophyllase gene hadmassive accumulation of the enzyme's product, chlorophyllide (Benedettiand Arruda, 2002). These plants retained the ability to synthesizechlorophyll and hence retained a green color.

Plants, because of their sessile nature, have evolved sophisticatedmechanisms for sensing and responding to their environment andsubstances in their environment (Trewavas, 2000, 2002). The presence ofa plant cell wall, now understood to be a complex matrix, does not deterthe ability of green plants to detect analytes (Dangl et al., 2000).Indeed, plants are capable of detecting analytes intracellularly (e.g.,soluble or cytoplasmic analytes such as chemicals or phytohormones) orextracellularly (e.g., certain pathogens, chemicals and gaseous hormonessuch as ethylene). Normal cytoplasmic analytes of plants are detectedwith a variety of receptors (Fujisawa et al., 2001; Friml et al., 2002)whereas normal extracellular analytes are sensed with membrane receptors(Dangl and Jones, 2001).

Sensing substances and linking the sensing to a response were recentlydeveloped in bacterial systems (Looger et al., 2003; Hellinga et al.,1991). These studies and related (Swartz J R, 2001; Allert et al., 2004;US Patent Application Publications 2004/229290 and 2004/0118681 and U.S.Pat. Nos. 6,977,180 and 6,521,446) show that (1) sensor or receptorproteins can, for a substance of interest, be designed in a computer and(2) the binding of the specific substance to the computationallydesigned receptor can be linked to a bacterial transcriptional responsesystem. Receptors for the bacterial system that have been designedinclude ones for an organophosphate surrogate of the nerve agent soman,heavy metals such as Zn²⁺, explosives such as TNT, herbicides such asglyphosate, and environmental pollutants such as MTBE.

There is a need in the art for monitoring systems characterized by fastfeedback, ability to reset, capacity for remote evaluation, low cost toallow widespread use, ease of public recognition and the ability foroperation and assessment without technically sophisticated operators orequipment.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

In one aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a sensor protein, said protein comprising a secretorysequence for directing the protein to the extracellular space of a plantcell and a binding region specific for a target substance of interest,wherein said protein undergoes a conformational change when the targetsubstance is bound.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a sensor protein, said protein comprising a secretorysequence for directing the protein to the extracellular space of a plantcell and a binding region specific for a target substance of interest,wherein said protein undergoes a conformational change when the targetsubstance is bound, wherein the target substance is a nerve gas, a heavymetal, a poison, a pollutant, a toxin, an herbicide, a polycyclicaromatic hydrocarbon, a benzene, a toluene, a xylene, a halogenated(chloro, fluoro, and chlorofluoro) hydrocarbon, a steroid or otherhormone, an explosive, or a degradation product of one of the foregoingcompounds.

In a further aspect of the present invention, a DNA construct isprovided comprising a plant operable promoter operably linked to anucleic acid sequence encoding a sensor protein, said protein comprisinga secretory sequence for directing the protein to the extracellularspace of a plant cell and a binding region specific for a targetsubstance of interest, wherein said protein undergoes a conformationalchange when the target substance is bound, wherein the target substanceis a nerve gas, a heavy metal, a poison, a pollutant, a toxin, anherbicide, a polycyclic aromatic hydrocarbon, a benzene, a toluene, axylene, a halogenated (chloro, fluoro, and chlorofluoro) hydrocarbon, asteroid or other hormone, an explosive, or a degradation product of oneof the foregoing compounds, and wherein the encoded sensor proteinspecifically binds trinitrotoluene.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a sensor protein, said protein comprising a secretorysequence for directing the protein to the extracellular space of a plantcell and a binding region specific for a target substance of interest,wherein said protein undergoes a conformational change when the targetsubstance is bound, wherein the target substance is a nerve gas, a heavymetal, a poison, a pollutant, a toxin, an herbicide, a polycyclicaromatic hydrocarbon, a benzene, a toluene, a xylene, a halogenated(chloro, fluoro, and chlorofluoro) hydrocarbon, a steroid or otherhormone, an explosive, or a degradation product of one of the foregoingcompounds, wherein the encoded sensor protein specifically bindstrinitrotoluene, and wherein the DNA construct comprises the sequence ofSEQ ID NO:8.

In one aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a protein which comprises the following domains: aplasma membrane targeting signal sequence, an extracellular domain forbinding a sensor protein, a transmembrane domain and a histidine kinasedomain for phosphorylating a protein with nuclear shuttling ortranscriptional activating functions, wherein the histidine kinase isactivated when a sensor protein binds to the extracellular domain.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a protein which comprises the following domains: aplasma membrane targeting signal sequence, an extracellular domain forbinding a sensor protein, a transmembrane domain and a histidine kinasedomain for phosphorylating a protein with nuclear shuttling ortranscriptional activating functions, wherein the histidine kinase isactivated when a sensor protein binds to the extracellular domain, andwherein the extracellular domain, the transmembrane domain and thehistidine kinase domain are derived from one or more bacterial genes andthe membrane targeting signal sequence is derived from a plant gene.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a sensor protein, said protein comprising a secretorysequence for directing the protein to the extracellular space of a plantcell and a binding region specific for a target substance of interest,wherein said protein undergoes a conformational change when the targetsubstance is bound, and wherein the secretory sequence is from PEX(Pollen Extension-like protein).

In yet another aspect of the present invention, a DNA construct isprovided comprising a plant operable promoter operably linked to anucleic acid sequence encoding a protein which comprises the followingdomains: a plasma membrane targeting signal sequence, an extracellulardomain for binding a sensor protein, a transmembrane domain and ahistidine kinase domain for phosphorylating a protein with nuclearshuttling or transcriptional activating functions, wherein the histidinekinase is activated when a sensor protein binds to the extracellulardomain, wherein the extracellular domain, the transmembrane domain andthe histidine kinase domain are derived from one or more bacterial genesand the membrane targeting signal sequence is derived from a plant gene,and wherein the membrane targeting signal sequence is from FLS2.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a protein which comprises the following domains: aplasma membrane targeting signal sequence, an extracellular domain forbinding a sensor protein, a transmembrane domain and a histidine kinasedomain for phosphorylating a protein with nuclear shuttling ortranscriptional activating functions, wherein the histidine kinase isactivated when a sensor protein binds to the extracellular domain, andwherein said histidine kinase domain comprises segments derived from abacterium and a plant.

In a further aspect of the present invention, a DNA construct isprovided comprising a plant operable promoter operably linked to anucleic acid sequence encoding a protein which comprises the followingdomains: a plasma membrane targeting signal sequence, an extracellulardomain for binding a sensor protein, a transmembrane domain and ahistidine kinase domain for phosphorylating a protein with nuclearshuttling or transcriptional activating functions, wherein the histidinekinase is activated when a sensor protein binds to the extracellulardomain, wherein said histidine kinase domain comprises segments derivedfrom a bacterium and a plant, and wherein said histidine kinase domaincomprises segments from a bacterial histidine kinase and a plant AHK4(Arabidopsis histidine kinase) protein.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a protein which comprises the following domains: aplasma membrane targeting signal sequence, an extracellular domain forbinding a sensor protein, a transmembrane domain and a histidine kinasedomain for phosphorylating a protein with nuclear shuttling ortranscriptional activating functions, wherein the histidine kinase isactivated when a sensor protein binds to the extracellular domain,wherein the extracellular domain, the transmembrane domain and thehistidine kinase domain are derived from one or more bacterial genes andthe membrane targeting signal sequence is derived from a plant gene, andwherein the sequence encoding the histidine kinase is that of SEQ IDNO:9.

In one aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a detectable marker or a response gene, wherein thepromoter is responsive to an internal signal caused by an externaltarget substance of interest, and wherein said detectable marker isexpressed when an external target substance of interest is bound to asensor protein.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a detectable marker or a response gene, wherein thepromoter is responsive to an internal signal caused by an externaltarget substance of interest, and wherein said detectable marker isexpressed when an external target substance of interest is bound to asensor protein, and wherein the detectable marker is a chlorophylldegradation enzyme or a functional fragment thereof.

In a further aspect of the present invention, a DNA construct isprovided comprising a plant operable promoter operably linked to anucleic acid sequence encoding a detectable marker or a response gene,wherein the promoter is responsive to an internal signal caused by anexternal target substance of interest, and wherein said detectablemarker is expressed when an external target substance of interest isbound to a sensor protein, wherein the detectable marker is achlorophyll degradation enzyme or a functional fragment thereof, andwherein the chlorophyll degradation enzyme is selected from the groupconsisting of red chlorophyll catabolite reductase (RCCR), pheophorbidea oxygenase (PaO), or chlorophyllase.

In yet another aspect of the present invention, a DNA construct isprovided comprising a plant operable promoter operably linked to anucleic acid sequence encoding a detectable marker or a response gene,wherein the promoter is responsive to an internal signal caused by anexternal target substance of interest, and wherein said detectablemarker is expressed when an external target substance of interest isbound to a sensor protein, wherein the detectable marker is achlorophyll degradation enzyme or a functional fragment thereof, furthercomprising a plant operable promoter responsive to an external targetsubstance of interest operably linked to a nucleic acid sequenceencoding an interfering RNA molecule specific for a chlorophyllbiosynthesis coding sequence.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a detectable marker or a response gene, wherein thepromoter is responsive to an internal signal caused by an externaltarget substance of interest, and wherein said detectable marker isexpressed when an external target substance of interest is bound to asensor protein, wherein the detectable marker is a chlorophylldegradation enzyme or a functional fragment thereof, further comprisinga plant operable promoter responsive to an external target substance ofinterest operably linked to a nucleic acid sequence encoding aninterfering RNA molecule specific for a chlorophyll biosynthetic enzymecoding sequence, and wherein the chlorophyll biosynthesis codingsequence encodes chlorophyll synthetase, protochlorophyllideoxidoreductase (POR) or genome uncoupling 4 (GUN4), a gene regulatingchlorophyll biosynthesis.

In a further aspect of the present invention, a DNA construct isprovided comprising a plant operable promoter operably linked to anucleic acid sequence encoding a detectable marker or a response gene,wherein the promoter is responsive to an internal signal caused by anexternal target substance of interest, and wherein said detectablemarker is expressed when an external target substance of interest isbound to a sensor protein, wherein the detectable marker is achlorophyll degradation enzyme or a functional fragment thereof, andwherein the plant operable promoter comprises a PhoB binding sequence.

In another aspect of the present invention, a DNA construct is providedcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a detectable marker or a response gene, wherein thepromoter is responsive to an internal signal caused by an externaltarget substance of interest, and wherein said detectable marker isexpressed when an external target substance of interest is bound to asensor protein, wherein the detectable marker is a chlorophylldegradation enzyme or a functional fragment thereof, wherein the plantoperable promoter comprises a PhoB binding sequence, and wherein theplant operable promoter is set forth in SEQ ID NO:1.

In yet another aspect, a DNA construct is provided comprising a plantoperable promoter operably linked to a nucleic acid sequence encoding aplant operable transcriptional activator, wherein the transcriptionalactivator is activated when phosphorylated by a histidine kinase.

In another aspect, a DNA construct is provided comprising a plantoperable promoter operably linked to a nucleic acid sequence encoding aplant operable transcriptional activator, wherein the transcriptionalactivator is activated when phosphorylated by a histidine kinase, andwherein the plant operable transcriptional activator is that of SEQ IDNO:4.

In a further aspect, a DNA construct is provided comprising a plantoperable promoter operably linked to a nucleic acid sequence encoding aplant operable transcriptional activator, wherein the transcriptionalactivator is activated when phosphorylated by a histidine kinase, andwherein the plant operable transcriptional activator is that of SEQ IDNO:11.

In another aspect, a transgenic plant is provided comprising a) a firstDNA construct comprising a plant operable promoter operably linked to anucleic acid sequence encoding a sensor protein, said protein comprisinga secretory sequence for directing the protein to the extracellularspace of a plant cell and a binding region specific for a targetsubstance of interest, wherein said protein undergoes a conformationalchange when the target substance is bound, and b) a second DNA constructcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a protein which comprises the following domains: aplasma membrane targeting signal sequence, an extracellular domain forbinding a sensor protein, a transmembrane domain and a histidine kinasedomain for phosphorylating a protein with nuclear shuttling ortranscriptional activating functions, wherein the histidine kinase isactivated when a sensor protein binds to the extracellular domain, andc) a third DNA construct comprising a plant operable promoter operablylinked to a nucleic acid sequence encoding a detectable marker or aresponse gene, wherein the promoter is responsive to the transcriptionalactivator protein, and wherein the detectable marker is expressed whenan external target substance of interest is bound to a sensor protein.

In yet a further aspect, the target substance of the DNA construct ofthe transgenic plant is a nerve gas, a heavy metal, a poison, apollutant, a toxin, an herbicide, a polycyclic aromatic hydrocarbon, abenzene, a toluene, a xylene, a halogenated (chloro, fluoro, andchlorofluoro) hydrocarbon, a steroid or other hormone, an explosive, ora degradation product of one of the foregoing compounds.

In another aspect, the encoded sensor protein of the DNA construct ofthe transgenic plant specifically binds trinitrotoluene.

In a further aspect, the DNA construct of the transgenic plant comprisesthe sequence of SEQ ID NO:8.

In another aspect, the extracellular domain, the transmembrane domainand the histidine kinase domain of the DNA construct of the transgenicplant are derived from one or more bacterial genes, and the membranetargeting signal sequence is derived from a plant gene.

In another aspect, the secretory sequence of the sensor protein of theDNA construct of the transgenic plant is from PEX.

In yet another aspect, the membrane targeting signal sequence of the DNAconstruct of the transgenic plant is from FLS2.

In another aspect, the histidine kinase domain of the DNA construct ofthe transgenic plant comprises segments derived from a non-plantorganism.

In another aspect, the histidine kinase domain of the DNA construct ofthe transgenic plant comprises segments derived from a non-plantorganism and a plant.

In a further aspect, the histidine kinase domain of the DNA construct ofthe transgenic plant comprises segments derived from a non-plantorganism and a plant.

In another aspect, the sequence encoding the histidine kinase of the DNAconstruct of the transgenic plant is that of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:10 or SEQ ID NO:12.

In yet another aspect, the detectable marker of the DNA construct of thetransgenic plant is a chlorophyll degradation enzyme or a functionalfragment thereof.

In a further aspect, a transgenic plant is provided with a detectablemarker wherein the plant loses detectable green color when thedetectable marker is expressed.

In another aspect, the chlorophyll degradation enzyme of the DNAconstruct of the transgenic plant is selected from the group consistingof red chlorophyll catabolite reductase (RCCR), pheophorbide a oxygenase(PaO), or chlorophyllase.

In a further aspect, a transgenic plant is provided comprising a plantoperable promoter responsive to a transcription activator proteinoperably linked to a nucleic acid sequence encoding an interfering RNAmolecule specific for a chlorophyll biosynthesis coding sequence.

In yet another aspect, the chlorophyll biosynthesis coding sequence ofthe DNA construct of the transgenic plant encodes chlorophyllsynthetase, protochlorophyllide oxidoreductase (POR) or GUN4, a generegulating chlorophyll biosynthesis.

In another aspect, the plant operable promoter of the DNA construct ofthe transgenic plant comprises a PhoB binding sequence.

In a further aspect, the plant operable promoter of the DNA construct ofthe transgenic plant is set forth in SEQ ID NO:1.

In yet another aspect, a transgenic plant is provided which furthercomprises a fourth DNA construct comprising a nucleic acid encoding achlorophyll degradation enzyme or a functional fragment thereof operablylinked to a plant operable promoter responsive to the transcriptionactivator protein, and wherein said promoter is not in nature associatedwith said sequence encoding a chlorophyll degradation enzyme.

In another aspect, a transgenic plant is provided which furthercomprises a fourth NA construct comprising a plant operable promoteroperably linked to a nucleic acid sequence encoding a plant operabletranscriptional activator, wherein the transcriptional activator isactivated when phosphorylated by a histidine kinase.

In a further aspect, the detectable marker of the transgenic plant is afunctional RNA.

In yet another aspect, the functional RNA of the transgenic plant is aninterfering RNA molecule.

In another aspect, the functional RNA of the transgenic plant inhibitsexpression of a chlorophyll biosynthesis coding sequence.

In a further aspect, the chlorophyll biosynthesis coding sequence of thetransgenic plant encodes chlorophyll synthetase, protochlorophyllideoxidoreductase (POR) or GUN4, a gene regulating chlorophyllbiosynthesis.

In yet another aspect, the detectable marker of the transgenic plant isa chlorophyll degradation enzyme.

In another aspect, the chlorophyll degradation enzyme of the transgenicplant is red chlorophyll catabolite reductase (RCCR), pheophorbide aoxygenase (PaO), or chlorophyllase.

In a further aspect, the detectable marker of the transgenic plant is aβ-glucuronidase, a β-galactosidase or a green or yellow fluorescentprotein.

In yet another aspect, the transcription activator protein of the DNAconstruct of the transgenic plant comprises a response regulator domain.

In another aspect, the response regulator domain of the DNA construct ofthe transgenic plant is derived from PhoB.

In a further aspect, the transcription activator protein of the DNAconstruct of the transgenic plant is a PhoB:VP64 translational fusionprotein.

In yet another aspect, the sequence encoding the PhoB:VP64 protein ofthe DNA construct of the transgenic plant is given in SEQ ID NO:4, SEQID NO:5 SEQ ID NO:6 or SEQ ID NO:11.

In another aspect, the detectable marker of the transgenic plant is afunctional RNA which inhibits expression of a chlorophyll biosyntheticenzyme coding sequence.

In a further aspect, a transgenic plant is provided, wherein the plantloses green color due to inhibition of chlorophyll biosynthesis andenhanced breakdown of chlorophyll upon induction of a gene encoding achlorophyll degradation enzyme.

In yet another aspect, a transgenic plant is provided, wherein the plantloses green color due to inhibition of chlorophyll biosynthesis andenhanced breakdown of chlorophyll upon induction of a gene encoding achlorophyll degradation enzyme and wherein the enhanced breakdown ofchlorophyll is achieved by expressing at least one enzyme selected fromthe group consisting of red chlorophyll catabolite reductase (RCCR),pheide a oxygenase (PaO), and chlorophyllase.

In another aspect, a transgenic plant is provided, wherein the plantloses green color due to inhibition of chlorophyll biosynthesis andenhanced breakdown of chlorophyll upon induction of a gene encoding achlorophyll degradation enzyme and wherein the inhibition of chlorophyllbiosynthesis is achieved by inhibiting expression of at least one enzymeselected from the group consisting of protochlorophyllide oxidoreductase(POR), chlorophyll synthetase and GUN4.

In a further aspect, the inhibition of POR in the transgenic plant isachieved by producing an interfering RNA molecule that contains asequence derived from the coding sequence of POR.

In yet another aspect, a transgenic plant is provided, wherein the plantloses green color by inhibiting POR and stimulating RCCR andchlorophyllase.

In another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker.

In a further aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and wherein thedetectable marker is a functional RNA.

In yet another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, wherein thedetectable marker is a functional RNA, and wherein the functional RNA isan interfering RNA molecule.

In another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, wherein thedetectable marker is a functional RNA, and wherein the functional RNAinhibits expression of a chlorophyll biosynthesis coding sequence.

In a further aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, wherein thedetectable marker is a functional RNA, and wherein the functional RNAinhibits expression of a chlorophyll biosynthesis coding sequence, andwherein the chlorophyll biosynthesis enzyme is a chlorophyll synthetase,protochlorophyllide oxidoreductase (POR) or a GUN4, a gene regulatingchlorophyll biosynthesis.

In yet another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and wherein thedetectable marker is a chlorophyll degradation enzyme.

In another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and wherein thedetectable marker is a chlorophyll degradation enzyme, and wherein thechlorophyll degradation enzyme is red chlorophyll catabolite reductase(RCCR), pheophorbide a oxygenase (PaO), or chlorophyllase.

In a further aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and wherein thedetectable marker is a β-glucuronidase, a β-galactosidase or a green oryellow fluorescent protein.

In yet another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and whereinsaid transgenic plant comprises a) a first DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a sensor protein, said protein comprising a secretory sequencefor directing the protein to the extracellular space of a plant cell anda binding region specific for a target substance of interest, whereinsaid protein undergoes a conformational change when the target substanceis bound, and b) a second DNA construct comprising a plant operablepromoter operably linked to a nucleic acid sequence encoding a proteinwhich comprises the following domains: a plasma membrane targetingsignal sequence, an extracellular domain for binding a sensor protein, atransmembrane domain and a histidine kinase domain for phosphorylating aprotein with nuclear shuttling or transcriptional activating functions,wherein the histidine kinase is activated when a sensor protein binds tothe extracellular domain, and c) a third DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a detectable marker or a response gene, wherein the promoter isresponsive to the transcriptional activator protein, and wherein thedetectable marker is expressed when an external target substance ofinterest is bound to a sensor protein and wherein said transcriptionactivator protein is a PhoB protein or is derived from a PhoB protein.

In another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and whereinsaid transgenic plant comprises a) a first DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a sensor protein, said protein comprising a secretory sequencefor directing the protein to the extracellular space of a plant cell anda binding region specific for a target substance of interest, whereinsaid protein undergoes a conformational change when the target substanceis bound, and b) a second DNA construct comprising a plant operablepromoter operably linked to a nucleic acid sequence encoding a proteinwhich comprises the following domains: a plasma membrane targetingsignal sequence, an extracellular domain for binding a sensor protein, atransmembrane domain and a histidine kinase domain for phosphorylating aprotein with nuclear shuttling or transcriptional activating functions,wherein the histidine kinase is activated when a sensor protein binds tothe extracellular domain, and c) a third DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a detectable marker or a response gene, wherein the promoter isresponsive to the transcriptional activator protein, and wherein thedetectable marker is expressed when an external target substance ofinterest is bound to a sensor protein and wherein said transcriptionactivator protein is a PhoB:VP64 translational fusion protein.

In a further aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and whereinsaid transgenic plant comprises a) a first DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a sensor protein, said protein comprising a secretory sequencefor directing the protein to the extracellular space of a plant cell anda binding region specific for a target substance of interest, whereinsaid protein undergoes a conformational change when the target substanceis bound, and b) a second DNA construct comprising a plant operablepromoter operably linked to a nucleic acid sequence encoding a proteinwhich comprises the following domains: a plasma membrane targetingsignal sequence, an extracellular domain for binding a sensor protein, atransmembrane domain and a histidine kinase domain for phosphorylating aprotein with nuclear shuttling or transcriptional activating functions,wherein the histidine kinase is activated when a sensor protein binds tothe extracellular domain, and c) a third DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a detectable marker or a response gene, wherein the promoter isresponsive to the transcriptional activator protein, and wherein thedetectable marker is expressed when an external target substance ofinterest is bound to a sensor protein and, wherein said sequenceencoding the PhoB:VP64 protein is given in SEQ ID NO:4 or SEQ ID NO:5.

In yet another aspect, a method is provided for detecting an externalsubstance of interest, said method comprising the step of exposing atransgenic plant to an external substance of interest and detecting achange resulting from expression of a detectable marker, and whereinsaid transgenic plant comprises a) a first DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a sensor protein, said protein comprising a secretory sequencefor directing the protein to the extracellular space of a plant cell anda binding region specific for a target substance of interest, whereinsaid protein undergoes a conformational change when the target substanceis bound, and b) a second DNA construct comprising a plant operablepromoter operably linked to a nucleic acid sequence encoding a proteinwhich comprises the following domains: a plasma membrane targetingsignal sequence, an extracellular domain for binding a sensor protein, atransmembrane domain and a histidine kinase domain for phosphorylating aprotein with nuclear shuttling or transcriptional activating functions,wherein the histidine kinase is activated when a sensor protein binds tothe extracellular domain, and c) a third DNA construct comprising aplant operable promoter operably linked to a nucleic acid sequenceencoding a detectable marker or a response gene, wherein the promoter isresponsive to the transcriptional activator protein, and wherein thedetectable marker is expressed when an external target substance ofinterest is bound to a sensor protein and wherein said change isdegreening of the transgenic plant.

In another aspect, the degreening of the transgenic plant is detectedvisually or by detecting properties selected from the group consistingof chlorophyll fluorescence, photosynthetic properties and propertiesrelated to reactive oxygen species and their damage.

In yet another aspect, the degreening of the transgenic plant isdetected by imaging selected from the group consisting of hyper-spectralimaging, infra-red imaging, near-infra-red imaging and multi-spectralimaging

In a further aspect, the transgenic plant regreens after removal of theexternal substance of interest.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than limiting.

FIGS. 1A-1E show the overview of the detection process and variousprotein components used in the sensing (or input) circuit and how theyare linked to a specific output. FIG. 1A: SS-TNT is an example of acomputer designed receptor (also referred to as sensor protein) based onbacterial periplasmic binding proteins. The protein leader for thebacteria is removed and replaced by a plant leader designated “SS”(secretory sequence), to target the sensor protein to the extracellularor apoplastic space. Any sensor protein, including but not limited to,trinitrotoluene (TNT), Methyl tert-butyl ether (MTBE), nerve gas,cyclotrimethylenetrinitramine (RDX) and those to be designed can beused. FIG. 1B shows a schematic of the ligand and computer designedreceptor (sensor proteins) (note that there are many receptors and thatonly one is shown). FIG. 1C shows that the sensor protein binds TNT orthe ligand (defined with computational design) with very high affinityand specificity. FIG. 1D shows that the sensor protein with the ligand(TNT) develops high affinity for the extracellular domain of thehistidine kinase (HK, Trg) and activates the histidine kinase domain.The histidine kinase is activated and high energy phosphate istransferred to another protein. In this figure, the shuttling protein ortranscription activator protein is PhoB:VP64. FIG. 1E shows thatPhoB:VP64 shuttles to the nucleus, binds the PlantPho promoter andactivates transcription of a linked gene or genes, in this case thedegreening circuit that produced white plants (or a lighter greencolor). FIG. 1F shows a schematic of the degreening circuit.

FIGS. 2A-2C show transmembrane proteins (histidine kinases) and theirpathways that are used for transcriptional response. FIG. 2A shows threehistidine kinases (HKs). In the first panel, after the receptors (sensorproteins) bind their ligand they develop a high affinity for Trg. HybridHKs containing Trg (light grey dashes) were formed and shown to functionin both bacteria and in plants. For plant expression, a protein leader,such as the FLS (dashed line) exemplified is used. This leader iscleaved during processing. Trg causes a conformational change activatingthe intracellular kinase. The high energy phosphate signal is thentransmitted by (left to right) the hybrid proteins AHK4, PhoR, or EnvZ.FIG. 2B shows the transmission protein for each hybrid HK shown in FIG.2A. FLS:Trg:EnvZ:AHK4 transmits its phosphate signal to histidinephosphotransferases (AHPs) whereas both FLS:Trg:PhoR and FIs:Trg:EnvZtransmit their phosphate signal to PhoB:VP64. FIG. 2C shows that bothproteins that receive a phosphate signal from the HKs translocate to thenucleus and activate expression of linked genes. The left panel shows,AHP translocates to the nucleus and activates expression of promoterssuch as ARR5 or ARR7 (Type A ARR promoters). The right panel showsPhoB:VP64 translocates to the nucleus and activates expression of asynthetic promoter PlantPho promoter.

FIGS. 3A-3E show evidence for signal-dependent nuclear shuttling ofbacterial response regulators in plants. FIG. 3A shows the bacterialresponse regulator (RR) PhoB translocates to the plant nucleus inresponse to an external cytokinin signal. FIG. 3A panels A-B showtransient assay of PhoB:GFP fusion protein in onion epidermal cells.Cells treated with cytokinin (B) had stronger nuclear PhoB:GFPaccumulation than control (A) cells. FIG. 3A panels C-P showsignal-dependent shuttling of PhoB:GFP to the nucleus in transgenicArabidopsis plants. Localization of PhoB:GFP in roots (C-H), in leaves(I-J, M-N), and the crown (K-L, O-P), a stem-like region. Prior tocytokinin treatment (C, I, K), fluorescence from PhoB:GFP is diffuse andthroughout the cells. After treatment with exogenous cytokinin (D-H, J,L-P), the same tissue shows distinct accumulation of PhoB:GFP incompartments that also stain with DAPI (F, H, N, P), indicating thatthey are nuclei. FIG. 3A panels Q-R show confocal microscopy of PhoB:GFProots showing that the protein enters the nucleus. Insets correspond toclose-up views. −CK, plant tissues prior to cytokinin treatment; +CKsame plant tissue after cytokinin treatment. DAPI, same tissues treatedwith DAPI to stain DNA. Arrowheads indicate nuclei. Scale bars=50 μm.FIG. 3B shows the bacterial response regulator (RR) OmpR translocates tothe plant nucleus in response to an external cytokinin signal. FIG. 3Bpanels A-D show signal-dependent nuclear localization of (OmpR:GFP inroots, panels E-J leaves, and panels K-N the crown, a stem-like region.Panels to the right correspond to close-up views of cells treated withcytokinin (+CK), or cells treated with cytokinin and DAPI forvisualization of nuclei (DAPI). FIG. 3B panels O-P show confocalmicroscopy of OmpR:GFP-expressing roots showing that the protein entersthe nucleus in response to the cytokinin signal. Insets correspond toclose-up views of cells. −CK, tissues incubated in the absence ofcytokinin +CK, tissues incubated in the presence of cytokinin; DAPI,indicate incubation with DAPI to stain DNA. Arrows point to cell nuclei.Scale bars=50 μm. FIG. 3C shows nuclear localization of PhoB:GFP:GUSfusion protein in response to the external cytokinin signal; panels B-Dshows that some type of transport mechanism must be involved in thenuclear shuttling. Nuclear identity is confirmed by DAPI staining panelE. Arrowheads point to cell nuclei. Scale bars=50 μm. FIG. 3D showsmutation of the phospho-accepting Asp residue in PhoB and OmpR disruptsshuttling. Upper panels, PhoB^(D53A):GFP or, lower panelsOmpR^(D55A):GFP, in transgenic plants. Some PhoB^(D53A):GFP localizes tothe base of root cortical cells (arrows); but nuclear shuttling is notseen in non-vascular cells. Some weaker nuclear localization is seen invascular cells (arrowheads). Mutation of the conserved Asp55 eliminatedshuttling of OmpR^(D55A):GFP. Arrowheads point to nuclei. −CK, plantsprior to cytokinin treatment; +CK same plant tissue after cytokinintreatment. DAPI, same tissues treated with DAPI to stain DNA. Scalebars=50 μm. FIG. 3E shows mutation of the phospho-accepting Asp residuein PhoB disrupts signaling from the TNT receptor. FIG. 3E panel A showsindependent transgenic plants containing the PhoB^(D53A):VP64mutagenized protein linked to the TNT sensing system were tested forinduction of GUS activity with 10 μM TNT. No significant induction wasobserved (levels were consistently less than 4, levels seen incontrols). FIG. 3E panel B shows a homozygous transgenic line containingthe wild-type PhoB:VP64 linked to the TNT sensing system consistentlyshows TNT induction.

FIGS. 4A-4D show data for function of computationally designed receptorsand hybrid histidine kinases in plants as measured by GUS activity. FIG.4A shows FLS:Trg:EnvZ:AHK4 signals to and activates plant AHPs,activating plant AHPs. Phosphorylated AHPs shuttle to the cell nucleus,where they transfer their phosphate group to a Type-B ARR. ActivatedType-B ARR then binds to and activates the ARR5 promoter. GUS activityis expressed as nmoles 4-MU mg⁻¹ protein h⁻¹. To, indicates primarytransformants; T1, indicates the next generation of transgenic plants.To P-value=0.0002; T1 P-value=0.0102. FIG. 4B shows FLS:Trg:PhoR signalsto PhoB:VP64, which translocates to the nucleus where it binds to andactivates the PlantPho promoter. FIG. 4C shows FLS:Trg:EnvZ (Trz)signals to PhoB:VP64, which translocates to the nucleus where it bindsto and activates the PlantPho promoter. GUS activity is expressed asnmoles 4-MU mg⁻¹ protein h⁻¹. FIG. 4D shows a variation of the VP64activation domain fused to PhoB (PhoB:VP16) also activates the PlantPhopromoter in response to an input (cytokinin in this case). GUS activityis expressed as nmoles 4-MU mg⁻¹ protein h⁻¹. CK, cytokinin.

FIGS. 5A-5F show function of the degreening circuits used as output.FIG. 5A upper panels show the appearance of two plants from eachdegreening circuit (number 1-5) at 0, 24 and 48 hours after induction.The middle panel shows a schematic for each degreening circuit.Represented is a chlorophyll molecule. The “stop” signs indicate genesthat were inhibited by an interfering RNA molecule (diRNA) construct;lightning bolts represent genes that were over-expressed. The bottompanel shows the appearance of plants that had been induced to fullydegreen and regreen for 3 days. FIG. 5B shows the results ofsemi-quantitative RT-PCR analysis of the degreening circuit transcriptsat 0, 24 and 48 hours after induction and regreening for 3 days. CYC,cyclophilins, was used as a sample loading control. In the controlsamples, −ind indicates samples without induction and +ind indicatessamples with induction. FIG. 5C shows remote monitoring of thedegreening circuit by effects on chlorophyll fluorescence. Time coursechanges of total chlorophyll and maximum chlorophyll fluorescence indark-adapted plants (Fm) (upper panel), operating efficiency of PSII(φPSII)(middle panel), and maximum quantum efficiency of PSII(Fv/Fm)(lower panel) in transgenic plants containing degreening circuit#1 and wild-type control (Columbia). Solid lines with filled squares,control plants; dashed lines with open squares, induced plants; dottedlines with open triangles, total chlorophyll levels of induced plants.F.W.: fresh weight. Plants were incubated in the presence (induced) orabsence (control) of the inducer. FIG. 5D shows induction of thedegreening results in accumulation of Reactive Oxygen Species (ROS).Auto-fluorescence from chlorophyll can be seen in both panels whereasROS is only seen in the degreening panel. FIG. 5E parts a-c, show rapidloss of chlorophyll upon induction of the degreening circuit requireslight both in whole plants and in detached leaves. FIG. 5E part a showsthe effect of light on degreening of whole plants. Fourteen-day oldplants containing degreening circuit #3 were induced to degreen andplaced in complete darkness. After 24, 48 or 72 hours, samples wereremoved from the dark, photographed, and transferred to normal lightconditions (approx. 100 μE·m−2.s−1; 16 h light/8 h dark). Plants werere-photographed after 24, 48 or 72 hours in light. Dark panels: plantsplaced in complete darkness. Arrows indicate samples moved to light.FIG. 5E part b shows degreening of detached leaves in the light fromplants containing different circuits. Top panels, leaves at 0 hours ofinduction; lower panels, leaves at 48 hours of induction. “C” indicatesthe Columbia control plants. 1, 2, and 3, indicate the specificdegreening circuit. FIG. 5E part c shows the effect of light ondegreening of detached leaves. Leaves were excised from 14-day oldplants containing circuit #2, and the degreening circuit was induced asdescribed above. Left panels show leaves incubated in the light; rightdark panels show leaves incubated in darkness; right lower panelsindicate leaves incubated in darkness after they were transferred tolight. Numbers next to panels indicate time of incubation, in hours.FIG. 5F shows the response of senescence-related genes duringdegreening. A list of 827 genes that are strongly and reproduciblyinduced during developmental leaf senescence (Buchanan-Wollaston et al.,2005) is used here as a basis for comparison with whole-genomemicroarray analysis of synthetic degreening (24h post-induction). Thedashed line in each plot corresponds to a degreening expression ratio of1.5 (0.58 log₂). Points above this line represent genes that areup-regulated at least 1.5 fold in both degreening and senescence. Pointsbelow the dashed line are strongly induced during senescence, but arenot strongly induced during degreening. Error bars show the standarderror of the mean expression ratio (degreening/uninduced). Genes areclassified in categories adapted from Buchanan-Wollaston et al., 2005.89% of the previously described senescence-induced genes were detectedin our microarray analysis; the remainder had signals that were too weakor variable to be accurately described. Minor categories with fewer thanfive members were combined with the genes of unknown function in plot(t).

FIGS. 6A-6D show functional plant sentinel via linking input todegreening circuit. TNT induced Degreening: Transgenic Arabidopsis orTobacco leaves containing ssTNT. FLS:Trg:PhoR, PhoB:VP64, and thePlantPho promoter controlling expression of the degreening genes, wereexcised and submerged in TNT. The progress of degreening was followed inone day increments. FIG. 6A shows induction of Arabidopsis degreeningwith 100 μM TNT. FIG. 6B shows induction of Tobacco degreening with 10μM TNT. FIGS. 6C-D show time-course monitoring of chlorophyllfluorescence as a measure of degreening in Arabidopsis and Tobaccodetached leaves induced to degreen. Chlorophyll measurements wereobtained using a Fluorcam. One of the parameters derived fromchlorophyll measurements is Fv/Fm, which is a measure of photosyntheticcapability (specifically maximum quantum yield of PSII). A normal leafwould have an F_(v)/F_(m) ratio of 0.8. Note the decline over time inthe F_(v)/F_(m) ratio as the leaves degreen, however the wild-typeColumbia leaf stayed close to 0.8. FIG. 6C shows Fluorcam data forArabidopsis T0 degreening. FIG. 6D shows Fluorcam data for Tobacco TOdegreening.

FIG. 7 shows a diagram of the function response circuit for floweringtime in plants. This system could also be used to control processesother than degreening. As exemplified in the diagram, the detection andsignaling systems could be linked to other outputs, for example, controlof flowering time in plants. Control of flowering time in response toTNT is used here. Furthermore, the input could also be changed, simplyby designing a new receptor (sensor protein), as detailed in thespecification. Genes that control flowering time in plants, such as FT(Flowering Locus T), SOC1 (Suppressor of overexpression of CO 1), andothers, could be placed under control of the PlantPho promoter. Thiswould provide an input-sensitive system to control flowering time.

FIG. 8 shows TNT induction of the degreening circuit independent oflight exposure. TNT-induced degreening of plants independent of lightcan be obtained by using the PlantPho promoter to direct expression ofone or more genes encoding chloroplast-localized proteases that areinvolved in turnover of the D1 reaction center protein. Examples ofproteases that could be used include, but are not limited to, DegP2,FtsH.

FIGS. 9A-9C show diagrams of the “trigger circuitry” for rapid inductionof the degreening circuit in response to a single exposure to an input(TNT exemplified). FIG. 9A shows setting the trigger circuitry to enableplants to respond to a single exposure to TNT. The degreening genes areunder control of a modified CaMV35S promoter. This modified CaMV35Spromoter contains the LexA DNA binding elements allowing Repressor 2 tobind and keep the degreening genes off. Expression of Repressor 2protein (LexA DBD/EAR2) is driven by a modified constitutive promoter(Pnos, weakly constitutive), containing DNA elements for the GAL4 DNAbinding domain. Plants functioning on the male side contain the normalsensing circuitry (TNT receptor, transmembrane HK, and PhoB:VP64), andtwo additional genes that both direct the expression of Repressor 1(containing the GAL4 DBD and EAR1 repressor domain). One gene directingthe expression of Repressor 1 is under the direction of the PlantPhopromoter. The second gene directing the expression of Repressor 1 isunder the direction of a weak constitutive promoter (Pnos) modified tocontain LexA DNA elements. To set the “trigger switch”, pollen obtainedfrom homozygous plants with the gene circuitry indicated for the maleplant is used to fertilize homozygous plants containing the genecircuitry indicated for the female plant. NLS, indicates nuclearlocalization sequence; nos, indicates 3′ terminator sequence from thenopaline synthase gene; ON, indicates the gene is expressed; OFF,indicates the gene is not expressed. FIG. 9B shows that in the absenceof the ligand (the explosive TNT) the receptors have no affinity for thetransmembrane histidine kinase, and the response regulator protein(PhoB:VP64) is unphosphorylated, causing its receiver domain to repressthe transactivation domain within this protein. The degreening genes aretranscriptionally off because constitutive expression of Repressor 2prevents expression of the (CaMV35S:LexA promoter. FIG. 9C shows inresponse to TNT, the receptor specifically binds the explosive anddevelops a high affinity for the histidine kinase (Trg/PhoR). Thiscauses a conformational change and activation of the kinase that thensends a high energy phospho-relay to PhoB:VP64. In the plants where the“trigger” genetic circuitry is set, phosphorylated PhoB:VP64translocates to the nucleus, binds the PlantPho promoter, and activatesexpression of Repressor 1. Repressor 1 then binds to and represses thePnos:GAL4 promoter. Repressing this promoter will turn OFF the Repressor2 protein (LexA/EAR2). Turning OFF the LexA/EAR2 protein allowsactivation of two genes: first, the degreening circuit genes (undercontrol of the CaMV 35S:LexA promoter); second, removing Repressor 2also allows expression of Repressor 1 under control of the Pnos:LexApromoter. Since the Pnos:LexA promoter does not need the continuousactivation from a phosphorylated PhoB:VP64, the detection system doesnot need continuous exposure to TNT. Therefore, expression of Pnos:LexAallows the readout (degreening) system to remain active even with asingle exposure to an explosive.

DETAILED DESCRIPTION OF THE INVENTION

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

The term, “transgenic plant”, is used herein to indicate a plant, orphotosynthetic organism including algae, which has been geneticallymodified to contain exogenous or heterologous DNA to obtain a desiredphenotype. Examples of the exogenous DNA molecules that have beentransformed into the plants of the invention include those encodingsegments of DNA encoding the sensor protein, the transmembrane protein,a shuttling and/or response protein, and a receptive promoter,collectively known as the response circuits and/or those encodingsegments of chlorophyll biosynthetic and/or complete degradation enzymesand a promoter which is responsive to a signal.

The term, “plant” as used in the present invention, is intended to coverany plant, vascular or nonvascular, aquatic or terrestrial; algae, andorganisms formally and informally recognized as algae now more properlyknown as cyanobacteria are included within this definition.

The term “non-plant organism” includes, but is not limited to Archea,bacteria, fungi including yeast and cyanobacteria and the like and otherorganisms containing two-component signaling systems.

The term “degreening”, also referred to as a “loss of green color”, isintended to indicate a loss of chlorophyll and photosynthetic pigmentsin the transgenic plants that is distinguishable from normal plants(non-transgenic plants). The degreening can be detected visibly, or witha variety of instruments that measure properties including but notlimited to chlorophyll fluorescence, hyper-spectral imaging, infra-redand near-infra-red imaging, multi-spectral imaging, photosyntheticproperties and properties related to reactive oxygen species and theirdamage. The measurement instruments can be hand-held, or instrumentsthat function at a distance, the distance being from aircraft orsatellites.

The term, “external signal”, or “environmental signal”, or “targetsubstance of interest”, is intended to mean a signal typically in theform of an analyte or ligand which triggers the signaling pathway in thetransgenic plants of the invention and results in the degreeningphenotype and/or change such as induction of gene expression ofinterest. In this sense, the signal can be any biological or chemicalagent including environmental pollutants. The substance can be, forexample, sugars, herbicides, a poison, a pollutant, a toxin, a nerve gassuch as soman, heavy metals such as mercury, lead, arsenic, uranium,cadmium, selenium, polycyclic aromatic hydrocarbon, a benzene, atoluene, a xylene, or a halogenated (chloro, fluoro, and chlorofluoro)hydrocarbon, a steroid or other hormone. In addition, the targetsubstance which binds to a specifically engineered input circuit via theextracellular receptor could be an explosive such as TNT(trinitrotoluene), RDX (cyclonite,hexahydro-1,3,5,-trinitro-1,3,5-triazine), HMX (octagen,octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) or TATP (triacetoneperoxide), or a degradation product of one of the foregoing compoundsrecognized by the input circuit via specific receptor site binding bythe sensor protein. Any target substance for which a sensor protein canbe computationally designed (Looger et al., 2003; Dwyer et al., 2004;Marvin and Hellinga, 2001) can serve as an external signal in thecontext of the present invention.

The term “detectable marker” is a change brought about in the plant thatis perceivable or capable of being sensed by humans, other organismssuch as but not limited to dogs, and/or machines. The change can bevisible or invisible to humans. The sensing can involve non-destructive(for example, multi-spectral imaging) or destructive methods (forexample, analysis of protein, DNA, RNA or metabolic product).

The term “response regulator domain” is a protein or portion of aprotein that contains conserved amino acids collectively functioning toperceive a phosphor-relay from an activated histidine kinase. Theconserved domain may contain a phosphor-accepting Asp or His residue orit may contain other residues that can be made capable of accepting theactivated phosphate.

The term “response gene” is a gene whose expression is linked to inputfrom the sensor protein or proteins.

The term “sensor protein” is used interchangeably with “receptor”.

The term “transmembrane protein” is used interchangeably with “histidinekinase”.

The terms “expression construct” or “DNA construct” are usedinterchangeably herein and indicate a DNA construct comprisingparticular sequences necessary for transcription of an associateddownstream sequence. An expression vector is a plasmid containing anexpression construct. If appropriate and desired for the associatedsequence, the term expression also encompasses translation (proteinsynthesis) of the transcribed RNA. The particular sequences contained inthe expression vector include a promoter, enhancer, termination signal,transcriptional block (Padidam, M and Cao, Y, 2001) and the like. Toprevent transcriptional interference from multiple transgenes, atranscriptional block is placed between appropriate genes on a planttransformation plasmid. A promoter is a DNA region which includessequences sufficient to cause transcription of an associated(downstream) sequence. The promoter may be regulated, i.e., notconstitutively acting to cause transcription of the associated sequence.If inducible, there are sequences present therein which mediateregulation of expression so that the associated sequence is transcribedonly when an inducer molecule is present. In the present context, theinducer molecule is analogous to the signal transmitted by an inputcircuit.

The term “derived from” includes genes, nucleic acids, and proteins whenthey include fragments or elements assembled in such a way that theyproduce a functional unit. The fragments or elements can be assembledfrom multiple organisms provided that they retain evolutionarilyconserved function. Elements or domains could be assembled from variousorganisms and/or synthesized partially or entirely, provided that theyretain evolutionarily conserved function, elements or domains. In somecases the derivation could include changes so that the codons areoptimized for expression in a particular organism.

The amino acids which occur in the various amino acid sequences referredto in the specification have their usual three- and one-letterabbreviations routinely used in the art: A, Ala, Alanine; C, Cys,Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe,Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, Ile, Isoleucine;K, Lys, Lysine; L, Leu, Leucine; M, Met, Methionine; N, Asn, Asparagine;P, Pro, Proline; Q, Gln, Glutamine; R, Arg, Arginine; S, Ser, Serine; T,Thr, Threonine; V, Val, Valine; W, Trp, Tryptophan; Y, Tyr, Tyrosine.

A protein is considered an isolated protein if it is a protein isolatedfrom a host cell in which it is recombinantly produced. It can bepurified or it can simply be free of other proteins and biologicalmaterials with which it is associated in nature.

One DNA portion or sequence is downstream of a second DNA portion orsequence when it is located 3′ of the second sequence. One DNA portionor sequence is upstream of a second DNA portion or sequence when it islocated 5′ of that sequence. Alternatively, the DNA sequences can bearranged in a functional polycistronic arrangement (Walker, J. M. andVierstra, R. D., 2007). Polycistronic messenger RNAs (mRNAs) are thoseresulting from transcription of two or more open reading frames (ORFs)fused together as one single mRNA from one promoter. A plant operablepolycistronic mRNA may be obtained by fusing a ubiquitin moiety betweentwo ORFs.

One DNA molecule or sequence is heterologous to another if the two arenot derived from the same ultimate natural source. The sequences may benatural sequences, or at least one sequence can be designed by man, asin the case of a multiple cloning site region or an entirely syntheticDNA sequence that encodes a gene or a fragment of a gene. The twosequences can be derived from two different species or one sequence canbe produced by chemical synthesis provided that the nucleotide sequenceof the synthesized portion was not derived from the same organism as theother sequence.

A polynucleotide is said to encode a polypeptide if, in its native stateor when manipulated by methods known to those skilled in the art, it canbe transcribed and/or translated to produce the polypeptide or afragment thereof. The anti-sense strand of such a polynucleotide is alsosaid to encode the sequence.

A nucleotide sequence is operably linked when it is placed into afunctional relationship with another nucleotide sequence. For instance,a promoter is operably linked to a coding sequence if the promotereffects the transcription or expression of the coding sequence.Generally, operably linked means that the sequences being linked arecontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. However, it is well known that certaingenetic elements, such as enhancers, may be operably linked even at adistance, i.e., even if not contiguous.

The term recombinant polynucleotide refers to a polynucleotide which ismade by the combination of two otherwise separated segments of sequenceaccomplished by the artificial manipulation of isolated segments ofpolynucleotides by genetic engineering techniques or by chemicalsynthesis. In so doing one may join together polynucleotide segments ofdesired functions to generate a desired combination of functions.

Large amounts of polynucleotides may be produced by replication in asuitable host cell. Natural or synthetic DNA fragments coding for aprotein of interest are incorporated into recombinant polynucleotideconstructs, typically DNA constructs, capable of introduction into andreplication in a prokaryotic or eukaryotic cell, such as Arabidopsisthaliana, wherein protein expression is desired. In addition to theArabidopsis thaliana specifically exemplified herein, other plants canbe used. Usually the construct is suitable for replication in aunicellular host, such as a bacterium, but a multicellular eukaryotichost may also be appropriate, with or without integration within thegenome of the host cell. Desirably, the DNA construct of interest isstably incorporated within the genome of a plant cell of interest forthe production of a sentinel plant for environmental monitoring.Commonly used prokaryotic hosts include strains of Escherichia coli,although other prokaryotes, such as Bacillus subtilis or a Pseudomonas,may also be used. Eukaryotic host cells include yeast, filamentousfungi, plant, insect, amphibian and avian species.

Polynucleotides may also be produced by chemical synthesis, e.g., by thephosphoramidite method described by Beaucage and Caruthers (1981) Tetra.Letts. 22: 1859-1862 or the triester method according to Matteuci et al.(1981) J. Am. Chem. Soc. 103: 3185, and may be performed on commercialautomated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

DNA constructs prepared for introduction into a prokaryotic oreukaryotic host typically comprise a replication system (i.e. vector)recognized by the host, including the intended DNA fragment encoding thedesired polypeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide-encoding segment. Expression systems (expression vectors)may include, for example, an origin of replication or autonomouslyreplicating sequence (ARS) and expression control sequences, a promoter,an enhancer and necessary processing information sites, such asribosome-binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and mRNA stabilizing sequences.Signal peptides may also be included where appropriate from secretedpolypeptides of the same or related species, which allow the protein tocross and/or lodge in cell membranes or be secreted from the cell.

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host. Examples of workablecombinations of cell lines and expression vectors are described inSambrook et al. (1989) vide infra; Ausubel et al. (Eds.) (1995) CurrentProtocols in Molecular Biology, Greene Publishing and WileyInterscience, New York; and Metzger et al. (1988) Nature, 334: 31-36.Many useful vectors for expression in bacteria, yeast, fungal,mammalian, insect, plant or other cells are well known in the art andmay be obtained from vendors such as Stratagene, New England Biolabs,Promega Biotech, CAMBIA and others. In addition, the construct may bejoined to an amplifiable gene so that multiple copies of the gene may bemade. For appropriate enhancer and other expression control sequences,see also Enhancers and Eukaryotic Gene Expression, Cold Spring HarborPress, N.Y. (1983). While such expression vectors may replicateautonomously, they may less preferably replicate by being inserted intothe genome of the host cell.

Expression and cloning vectors will likely contain a selectable marker,that is, a gene encoding a protein necessary for the survival or growthof a host cell transformed with the vector. Although such a marker genemay be carried on another polynucleotide sequence co-introduced into thehost cell, it is most often contained on the cloning vector. Only thosehost cells into which the marker gene has been introduced will surviveand/or grow under selective conditions. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxicsubstances, e.g., ampicillin, neomycin, methotrexate, kanamycin,hygromycin, BASTA, glyphosate, etc.; (b) complement auxotrophicdeficiencies; or (c) supply critical nutrients not available fromcomplex media. The choice of the proper selectable marker will depend onthe host cell; appropriate markers for different hosts are known in theart.

Recombinant host cells, in the present context, are those which havebeen genetically modified to contain an isolated DNA molecule. The DNAcan be introduced by any means known to the art which is appropriate forthe particular type of cell, or plant or eukaryotic organisms, includingwithout limitation, Agrobacterium-mediated, bacterial mediated,transformation, lipofection, particle bombardment or electroporation.

It is recognized by those skilled in the art that the DNA sequences mayvary due to the degeneracy of the genetic code and codon usage.

Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primedsynthesis of a nucleic acid sequence. This procedure is well known andcommonly used by those skilled in this art (see Mullis, U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science230:1350-1354). PCR is based on the enzymatic amplification of a DNAfragment of interest that is flanked by two oligonucleotide primers thathybridize to opposite strands of the target sequence. The primers areoriented with the 3′ ends pointing towards each other. Repeated cyclesof heat denaturation of the template, annealing of the primers to theircomplementary sequences, and extension of the annealed primers with aDNA polymerase result in the amplification of the segment defined by the5′ ends of the PCR primers. Since the extension product of each primercan serve as a template for the other primer, each cycle essentiallydoubles the amount of DNA template produced in the previous cycle. Thisresults in the exponential accumulation of the specific target fragment,up to several million-fold in a few hours. By using a thermostable DNApolymerase such as the Taq polymerase, which is isolated from thethermophilic bacterium Thermus aquaticus, the amplification process canbe completely automated. Other enzymes which can be used are known tothose skilled in the art.

It is well known in the art that the polynucleotide sequences of thepresent invention can be truncated and/or mutated such that certain ofthe resulting fragments and/or mutants of the original full-lengthsequence can retain the desired characteristics of the full-lengthsequence. A wide variety of restriction enzymes which are suitable forgenerating fragments from larger nucleic acid molecules are well known.In addition, it is well known that Bal31 exonuclease can be convenientlyused for time-controlled limited digestion of DNA. See, for example,Maniatis (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, pages 135-139, incorporated herein byreference. See also Wei et al. (1983 J. Biol. Chem. 258:13006-13512. Byuse of Bal31 exonuclease (commonly referred to as “erase-a-base”procedures), the ordinarily skilled artisan can remove nucleotides fromeither or both ends of the subject nucleic acids to generate a widespectrum of fragments which are functionally equivalent to the subjectnucleotide sequences. One of ordinary skill in the art can, in thismanner, generate hundreds of fragments of controlled, varying lengthsfrom locations all along a starting nucleotide sequence. The ordinarilyskilled artisan can routinely test or screen the generated fragments fortheir characteristics and determine the utility of the fragments astaught herein. It is also well known that the mutant sequences of thefull length sequence, or fragments thereof, can be easily produced withsite directed mutagenesis. See, for example, Larionov, O. A. andNikiforov, V. G. (1982) Genetika 18(3):349-59; Shortle, D., DiMaio, D.,and Nathans, D. (1981) Annu. Rev. Genet. 15:265-94; both incorporatedherein by reference. The skilled artisan can routinely producedeletion-, insertion-, or substitution-type mutations and identify thoseresulting mutants which contain the desired characteristics of the fulllength wild-type sequence, or fragments thereof, i.e., those whichretain promoter activity. It is well known in the art that there are avariety of other PCR-mediated methods, such as overlapping PCR that maybe used.

“Expression control sequences” are DNA sequences involved in any way inthe control of transcription or translation. Suitable expression controlsequences and methods of making and using them are well known in theart. The expression control sequences must include a promoter. Thepromoter may be any DNA sequence which shows transcriptional activity inthe chosen plant cells, plant parts, or plants. The promoter may beinducible or constitutive. It may be naturally-occurring, may becomposed of portions of various naturally-occurring promoters, or may bepartially or totally synthetic. Guidance for the design of promoters isprovided by studies of promoter structure, such as that of Harley andReynolds, Nucleic Acids Res., 15, 2343-61 (1987). Also, the location ofthe promoter relative to the transcription start may be optimized. Manysuitable promoters for use in plants are well known in the art as arenucleotide sequences which enhance expression of an associatedexpressible sequence.

For instance, suitable constitutive promoters for use in plants includepromoters from plant viruses, such as the peanut chlorotic streakcaulimovirus (PCISV) promoter (U.S. Pat. No. 5,850,019), the 35Spromoter from cauliflower mosaic virus (CaMV) (Odell et al., Nature313:810-812 (1985)), promoters of Chlorella virus methyltransferasegenes (U.S. Pat. No. 5,563,328), and the full-length transcript promoterfrom figwort mosaic virus (FMV) (U.S. Pat. No. 5,378,619); the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)), ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)), pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)), MAS (Velten etal., EMBO J. 3:2723-2730 (1984)), maize H3 histone (Lepetit et al., Mol.Gen. Genet. 231:276-285 (1992) and Atanassova et al., Plant Journal2(3):291-300 (1992)), Brassica napus ALS3 (WO 97/41228); and promotersof various Agrobacterium genes (see U.S. Pat. Nos. 4,771,002, 5,102,796,5,182,200 and 5,428,147). Finally, promoters composed of portions ofother promoters and partially or totally synthetic promoters can beused. See, e.g., Ni et al., Plant J., 7:661-676 (1995) and WO 95/14098describing such promoters for use in plants.

The promoter may include, or be modified to include, one or moreenhancer elements. Preferably, the promoter will include a plurality ofenhancer elements. Promoters containing enhancer elements provide forhigher levels of transcription as compared to promoters that do notinclude them. Suitable enhancer elements for use in plants include thePCISV enhancer element (U.S. Pat. No. 5,850,019), the CaMV 35S enhancerelement (U.S. Pat. Nos. 5,106,739 and 5,164,316) and the FMV enhancerelement (Maiti et al., Transgenic Res. 6:143-156 (1997)). See also WO96/23898 and Enhancers And Eukaryotic Expression (Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1983).

A 5′ untranslated sequence is also employed. The 5′ untranslatedsequence is the portion of an mRNA which extends from the 5′ CAP site tothe translation initiation codon. This region of the mRNA is necessaryfor translation initiation in plants and plays a role in the regulationof gene expression. Suitable 5′ untranslated regions for use in plantsinclude those of alfalfa mosaic virus, cucumber mosaic virus coatprotein gene, and tobacco mosaic virus. It is understood that thereshould be a ribosome binding site (such as a Kozak sequence which is aDNA sequence which “surrounds” (both ends of) the ATG start signal (fortranslation of mRNA)) associated with the coding sequence on the mRNA.

For efficient expression, the coding sequences are preferably alsooperatively linked to a 3′ untranslated sequence. The 3′ untranslatedsequence will include a transcription termination sequence and apolyadenylation sequence. The 3′ untranslated region can be obtainedfrom the flanking regions of genes from Agrobacterium, plant viruses,plants or other eukaryotes. Suitable 3′ untranslated sequences for usein plants include, but are not limited to, those from the cauliflowermosaic virus 35S gene, the phaseolin seed storage protein gene, the pearibulose biphosphate carboxylase small subunit E9 gene, the soybean 7Sstorage protein genes, the octopine synthase gene, and the nopalinesynthase gene.

The term “RNA interfering molecule” includes but is not limited todiRNA, siRNA miRNA, or an antisense RNA to inhibit synthesis of arelated coding sequence. It is part of a mechanism for RNA-guidedregulation of gene expression in which double-stranded ribonucleic acid(RNA) inhibits the expression of genes with complementary nucleotidesequences.

As noted above, the DNA construct may be a vector. The vector maycontain one or more replication systems which allow it to replicate inhost cells. Self-replicating vectors include plasmids, cosmids and viralvectors. Alternatively, the vector may be an integrating vector whichallows the integration into the host cell's chromosome of the DNAconstruct encoding the chlorophyll degrading enzyme and/or thechlorophyll biosynthesis-inhibiting sequence. The vector desirably alsohas unique restriction sites for the insertion of DNA sequences. If avector does not have unique restriction sites, it may be modified tointroduce or eliminate restriction sites to make it more suitable forfurther manipulations. It may also contain recombination sites to allowa variety of genes or gene fragments to be assembled or disassembled,accordingly.

The DNA constructs of the invention can be used to transform any type ofplant or plant cell. A genetic marker can be used for selectingtransformed plant cells (“a selection marker”). Selection markerstypically allow transformed cells to be recovered by negative selection(i.e., inhibiting growth of cells that do not contain the selectionmarker) or by screening for a product encoded by the selection marker.The most commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) gene, isolated from Tn5,which, when placed under the control of plant expression controlsignals, confers resistance to kanamycin. Fraley et al., Proc. Natl.Acad. Sci. USA 80:4803 (1983). Another commonly used selectable markergene is the hygromycin phosphotransferase gene which confers resistanceto the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol.,5:299 (1985). Additional selectable marker genes of bacterial originthat confer resistance to antibiotics include gentamycin acetyltransferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyltransferase, and the bleomycin resistance determinant (Hayford et al.1988. Plant Physiol. 86:1216, Jones et al. 1987. Mol. Gen. Genet.210:86; Svab et al. 1990. Plant Mol. Biol. 14:197, Hille et al. 1986.Plant Mol. Biol. 7:171). Other selectable marker genes confer resistanceto herbicides such as glyphosate, glufosinate or bromoxynil (Comai etal. 1985. Nature 317:741-744, Stalker et al. 1988. Science 242:419-423,Hinchee et al. 1988. Bio/Technology 6:915-922, Stalker et al. 1988. J.Biol. Chem. 263:6310-6314, and Gordon-Kamm et al. 1990. Plant Cell2:603-618).

Additional selectable markers useful for plant transformation include,without limitation, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz et al. 1987. Somatic Cell Mol. Genet. 13:67, Shah etal. 1986. Science 233:478, Charest et al. 1990. Plant Cell Rep. 8:643;EP 154,204).

Commonly used genes for screening presumptively transformed cellsinclude, but are not limited to, β-glucuronidase (GUS), β-galactosidase,luciferase, and chloramphenicol acetyltransferase (Jefferson, R. A.1987. Plant Mol. Biol. Rep. 5:387, Teeri et al. 1989. EMBO J. 8:343,Koncz et al. 1987. Proc. Natl. Acad. Sci. USA 84:131, De Block et al.1984. EMBO J. 3:1681), green fluorescent protein (GFP) and its variants(Chalfie et al. 1994. Science 263:802, Haseloff et al. 1995. TIG11:328-329 and WO 97/41228). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway (Ludwig et al. 1990. Science 247:449). Anotherscreening method is to look for functional degreening phenotype asdescribed herein in response to a specific inducer. Allowing the plantto regreen independent of the inducer allows recovery of the transgenicline.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; and Ausubel et al. (1992) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature,where employed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein. Intermediate cloningof the PCR products can be done using the PCRTerminator end repair kitand CLoneSmart kit vector pSMART (Lucigen, Middleton, Wis.). Various PCRbased cloning methods were used and are known to those skilled in theart.

All references cited in the present application are incorporated byreference herein to the extent there is no inconsistency with thepresent disclosure. References cited herein reflect the level of skillin the relevant arts.

The present invention exploits plants' sensing mechanisms forextracellular signals, with the development of plants that respond to avariety of biological, chemical, and environmental pollutants forsubstances of interest to produce a readily detectable response orphenotype. In a particular embodiment the plants disclosed herein losegreen color when exposed to a specific substance; the degreening is aneasily detectable biomarker and does not require sophisticatedinstrumentation. These plants function as “sentinels” and are especiallyuseful for widespread monitoring of substances in the environmentwhether interior or exterior.

For the plants to be useful as degreening biomarkers to detect specificchemical agents or to monitor environmental factors, an appropriateinput circuit was produced. This input circuit is useful for linkingdetection to response. When the input circuit is linked to thedegreening circuit, a plant detector is produced. In addition, theability to control response of plants and biological organisms tospecific substances provides a useful tool for biotechnology allowing,for example, co-ordination of crop plants, facilitating harvesting andcontrolling other developmental, tissue or environmental responses.

The present invention provides a highly specific and sensitive methodfor plants to detect a target substance of interest in theirenvironment, transmit the sensing from outside the plant to the nucleus,induce a specific transcriptional response and a type of outputcontrolled degreening that provides detection to humans. In oneembodiment of the invention, the regulatory circuits have twocomponents, referred to herein as input and output circuits. In anotherembodiment of the invention, the input circuit has an ability tospecifically recognize (bind) the target substance of interest andtransmit a signal to the nucleus, where a specific response isinitiated. The response can be a phenotypic and/or metabolic change ofinterest or a visible response to produce a plant sentinel. In oneembodiment of the invention, one output circuit produces a degreening orother detectable phenotype in the transgenic plant containing thecircuits. In one embodiment of the invention, the output circuit is alsomodular in that a variety of genes can be placed under control of thesignal-inducible promoter. In one embodiment of the invention, the inputcircuit of the invention is modular in that the receptor that istargeted to the extracellular space can be designed to providespecificity and selectivity for binding a given target substance ofinterest. One specific input circuit specifically exemplified hereinprovides detection of the explosive trinitrotoluene. One specific outputcircuit specifically exemplified herein serves as a simple and sensitivemarker that can easily be recognized directly (visually), or by remotesensing and/or by monitoring changes in chlorophyll fluorescence, bychanges in photosystem I and/or photosystem II, electron transport, bychanges in hyper-spectral imaging, and/or by changes in spectralproperties.

The input circuit comprises a sensor protein specifically targeted tothe extracellular space of the plant with a binding site specific forrecognizing a target agent or a target substance of interest, atransmembrane histidine kinase protein, a nuclear shuttling protein, anda synthetically designed signal responsive promoter. Variations andelaborations described herein are found in various researchpublications, and known to those skilled in the art. One type of outputcircuit described herein activates the expression of one or more genes,which results in a degreening phenotype in transgenic plants containingthe circuits.

The present invention provides a sensor protein or receptor at the cellsurface, such that the sensor protein or receptor has a binding sitespecific for the target substance of interest. The transmembraneprotein, a second component of the input circuit has three parts: aninteracting domain, a transmembrane domain and a histidine kinasedomain. Binding of the target substance of interest causes aconformational change in the sensor protein or receptor, so that it thenbinds to an interacting domain of the transmembrane protein on theexterior surface. The interaction of the sensor protein:target substanceof interest complex results in activation of the histidine kinase,typically by an autophosphorylation mechanism. The interaction of thesensor protein or receptor with the interacting domain produces aconformational change in the transmembrane protein and/or transmembranehistidine kinase. The autophosphorylated histidine kinase domain of thetransmembrane protein transfers a high energy phosphate group to acytoplasmically located protein. A variety of proteins will function inthe specific example described here: a synthetically adapted shuttlingprotein such as PhoB:VP64, other shuttling proteins such as histidinephosphotransferases, Arabidopsis histidine phosphotransferase, and othernatural proteins such as response regulators from plants, bacteria,fungi, and cyanobacteria systems, including adapted or syntheticproteins that function in histidine kinase mediated signaling systems.

The shuttling protein typically has several functions includingreception of the signal from the transmembrane protein, relay of thissignal to the nucleus, or specific responding component, and/oractivation of transcription. The protein may directly, or indirectly,bring about a cellular response. The typical cellular response isactivation of transcription, however, other responses are possibleincluding changes in membrane potential, cell expansion (in the case ofengineering a response that would allow expansion of the xylem), orchanges in the accumulation of a plant-derived product. At least someproteins are phosphorylated (directly) by the histidine kinase domain ofthe transmembrane protein. The phosphorylation of the proteins orprotein components can cause an increase in binding affinity for aspecific sequence of DNA as is the case for OmpR, or in the case ofPhoB, allow a conformational change that removes repression, allowingthe DNA binding domain to function. One type of response of this is areadout circuit that includes expression of the specifically regulatedgene located in the nucleus of the plant and the production of adetectable phenotype, appearance or function of lack thereof or thereadout can include activation of a gene controlling a trait ofinterest, for example, flowering or ripening.

The sensor protein or receptor can be derived from a bacterial (e.g.,Escherichia coli) periplasmic binding protein (PBP), such as a maltose,ribose or galactose PBP, and the binding site for the target substanceof interest can be a naturally occurring binding site or one which isthe result of computational design. At the N-terminus there is also asignal peptide sequence for targeting the sensor protein to the exteriorof the plant and plant cell; as specifically exemplified, the signalpeptide is that of the pollen PEX protein (Baumberger, N. et al., 2003).Substances of interest can include, without limitation, plant hormones,explosives, chemical agents such as a nerve agent (e.g., soman),environmental pollutants including all currently listed environmentalpollutants on the Environmental Protection Agency (EPA) superfund site,halogenated hydrocarbons, or degradation products, metal ions such aszinc, a heavy metal, a sugar, neurotransmitter, herbicides, pathogenicproducts, or an amino acid.

When the target substance of interest is bound to the sensor protein orreceptor, there is an interaction with the protein which transmits asignal from the exterior of the plant to a protein byautophosphorylation and activation of the histidine kinase. Upon bindingof the target substance of interest, there is an interaction between thesensor protein or receptor and the transmembrane protein (which containsthe histidine kinase domain). This interaction causesautophosphorylation of a histidine residue located on the transmembraneprotein. The phosphate is then transferred (a mechanism calledphosphor-relay or phosphotransfer) to a shuttling protein ortranscription activator protein domain, allowing it to translocate tothe nucleus or otherwise initiate a response. The phosphorylatedprotein, protein domain or secondary protein then binds a DNArecognition sequence present in a promoter of a gene (or genes) in thenucleus, which can be a genetically engineered gene, with the resultthat transcriptional expression of that gene occurs.

The transmembrane protein can be genetically engineered as atranslational fusion consisting of the plant and/or bacterial proteins,derived from one or more bacterial or plant proteins, derived from oneor more proteins containing histidine kinase-like features, orsynthetically synthesized features, provided that it functions in plantsin conjunction with a protein or protein domain to transmit the signalto a response unit. As specifically exemplified, it can be a translationfusion of FLS-TRZ (Trg-EnvZ) with AHK4 (see herein below), atranslational fusion of FLS-Trg-PhoR, or a translational fusion ofFLS-Trg-EnvZ. The intracellular receptive protein or protein domain canbe a plant protein, a bacterial protein or a synthetically designedprotein, with the proviso that it receives the signal from thetransmembrane protein. The receptive protein can either transmit thesignal to another protein that initiates a response or translocate tothe nucleus in response to the signal. In the case specificallyexemplified, the signal receptive protein itself moves to the nucleus,binds DNA and activates gene expression. As specifically exemplified, itcan be a plant histidine phosphotransferase or a bacterial protein suchas the E. coli proteins OmpR or preferably PhoB. Where the signalreceptive protein is also a transcriptional activation protein, PhoB,the DNA recognition sequence is CTGTCATAYAYCTGTCACAYYN (SEQ ID NO:14),and it can occur from 2 to 12 times, exemplified 4 or 8 times in theregion upstream of the transcription start site, and includes a planttranscriptional start site such as defined by a minimal transcriptionalpromoter.

The sequence which is expressed in response to detection of, or thepresence of, a target substance of interest in the plant environment canbe a protein coding sequence or it can be a functional nucleic acidsequence (such as a RNA interfering molecule, diRNA or an antisense RNAto inhibit synthesis of a related coding sequence) or it can be acombination of these. The associated expressed sequence can be a plantgene which is, in nature, expressed constitutively or in a tissue orcondition specific fashion, but in the present invention, it isexpressed when the target substance of interest or substance which bindsto the sensing protein or sensing proteins is present or after thetarget substance of interest is present. The expressed sequence can bevirtually any sequence of interest: a detectable marker such as green oryellow fluorescent protein or another fluorescent protein,β-glucuronidase or β-glucosidase, among others, a positive regulator offlowering or a sterility protein preferably selectively expressed in theappropriate tissue, a bioremediation coding sequence such as mercuryreductase, a phytochelatin or metal sequestering protein, an enzyme fordetoxifying a contaminant or harmful material, and the production of aspecific nutritive or pharmaceutical substance, among others. Theexpressed sequence can also be a functional nucleic acid (antisense ordiRNA to inhibit expression of a related nucleic acid sequence). Therecan be more than one target substance-regulated gene within a singleplant.

In an embodiment of the invention, the sensing circuitry can be used tocontrol features of interest such as, the timing of flowering of a plantor ripening of a fruit such that harvesting is more synchronized,coordination of crops such as cotton, soybean and corn and hence anability to predict harvest time, and thus, make harvesting moreefficient and economical or so that plants are in flower for aparticular occasion such as Easter, Mother's Day, Valentine's Day,Administrative Professional's Day or other holiday. Such a gene orresponse unit is operably linked to a promoter containing therecognition sequence of the specific sensing system or systems.

In another embodiment, the target substance of interest-dependenttranscription regulatory system can be used to render plants exposed tothe target substance sterile, when a sterility inducing protein isexpressed under the regulatory control of the control system of thepresent invention.

Within the scope of the present invention are one or more DNA constructscontaining a plant operable sensor protein as described above, a planttransmembrane protein, a plant operable signal reception and/ortranscription activation protein that is activated by the histidinekinase portion of the sensing circuit (via an intermediary endogenousprotein, the AHP, or directly by the membrane bound kinase), and a plantoperable sequence operably linked to transcription regulatory sequenceswhich include the recognition sequence of the particular transcriptionactivating protein of the invention.

Similarly, the present invention provides transgenic plant cells,transgenic plant parts, transgenic plant tissue and transgenic plantscontaining one or more constructs of the present invention.

The present invention provides transgenic (sentinel) plants useful forenvironmental monitoring and for detecting particular biological andchemical agents, environmental pollutants, and/or a specific substancesuch as herbicides or trigger compounds. Trigger compounds aresubstances that bind to the natural or computationally designed sensorproteins and thereby increase the sensor proteins affinity for anextracellular protein domain, as specifically exemplified herein, Trg.In a specific embodiment, the plants disclosed herein lose green colorwithin hours of exposure to particular target biological/chemical agentsor environmental pollutants. The loss of green color (or a change in thefluorescence of chlorophyll or a change in photosynthetic electrontransport) in plants is easily detectable, either by direct observation,with simple hand-held machines, or remotely by aircraft or satellitesensing. The sentinel plants of the present invention comprisegenetically engineered DNA constructs which direct the expression ofboth the input and output circuits, as described below, with the resultthat the plants lose color when they “sense” the presence of the targetsubstance of interest. An important advantage of the degreening systemin these sentinel plants is that they are capable of regreening. Theyeither regreen naturally or at an enhanced rate with treatment ofhormones, i.e., the sentinels can be reset for renewed surveillance forthe target substance to which they respond (Antunes et al., 2006). Inone aspect of the present invention, a transgenic plant whereindegreening has occurred due to the presence of a target substance ofinterest is able to regreen after removal of the external targetsubstance of interest.

The transgenic plants (sentinel plants) of the present invention can beindoor plants, for example, any of a number of species that are commonlyused as decorative accent plants, such as peace lily (Spathiphyllum),philodendron, pothos (Epipremnum), spider plant (Chlorophytum),Tradescantia and Dracaena, and the like. In addition, the sentinelplants can be crop plants such as corn, wheat, soy, cotton, soybeans andothers, or they can be grasses or trees, either deciduous (poplars,aspens, maple, oak, cottonwood, and the like) or evergreen (pines,spruce, junipers and the like) or they can be annuals or perennials usedin various types of plantings, or they can be a variety of nativespecies, or they can be aquatic plants including, but not limited to,algae. Nearly all plants and/or plant cells can be readily transformedand transformed seed directly formed or plants produced from thetransformed cells, as is well known to the art. The sentinel plants ofthe present invention can provide a warning of current presence of atarget substance of interest or they can provide notice to responders toa scene to allow for appropriate protective measures and/or to preventexposure to a dangerous condition. In addition, the sentinel plantsprovide the ability to remotely monitor for the presence of substances.Moreover, the sentinel plants allow for continuous environmentalmonitoring over extremely large scales (e.g., hundred or thousands ofsquare kilometers) that is not currently possible with any otherpublicly known method.

The sentinel plants of the invention contain a genetically engineeredsignaling pathway consisting of two functional parts referred to hereinas “input” and “output” wherein one embodiment of the output is the“degreening” circuit”. The input gene circuit is a natural orgenetically engineered system that recognizes a biological or chemicalagent, explosive, or an environmental pollutant or target substance ofinterest specifically and selectively, then activates an output genecircuit that results in the desired response. In the case of a plantsentinel, the output gene circuit shown here is the degreening circuit,so that the degreening phenotype i.e., white plants, are produced inresponse to an agent or pollutant. The degreening can be visuallydetected as a loss of green color or it can be detected as a change inchlorophyll fluorescence or in photosynthetic electron transport or itcan be detected with a variety of spectroscopic methods such ashyper-spectral imaging and other methods.

The output and input circuits of the invention are generated byexpressing DNA constructs specifically designed to provide a functionalsystem. The input circuit is a system comprising a receptor or a bindingprotein designed to recognize (e.g. by binding) a signal (e.g. analyteor ligand), and this binding event ultimately activates a response, oneof which is transcription of a gene of the output (degreening) circuitto produce a plant sentinel. Thus, the specificity and selectivity of agiven response is determined by the input circuit. An example of theinput circuit is a receptor or binding protein (sensor protein) whichspecifically binds a particular explosive, chemical agent or apollutant, the target substance of interest, which, upon binding of suchexplosive, agent or pollutant, can transmit a signal via thetransmembrane protein to activate transcription of a gene(s) in theoutput circuit. As specifically exemplified the sensor protein:targetsubstance complex interacts with the exterior domain of thetransmembrane protein, with the result that the histidine kinase becomesactive. A sensor protein or receptor specific for a given ligand oranalyte (target substance of interest), can be designed by usingcomputational design (Looger, et al., 2003; Dwyer, et al., 2003; Marvin,J S and Hellinga H W, 2001).

The response system (output, as exemplified by degreening) circuit isgenerated by transforming a plant with DNA constructs (i.e. expressionvectors) comprising one or more nucleic acids encoding, or complementaryto a nucleic acid encoding key enzymes or functional fragments thereofin chlorophyll biosynthesis and/or degradation pathway under the controlof a promoter which responds to a signal from the input circuit. Theterm “functional fragment” as used herein, is intended to indicate thatthe product (i.e., enzyme) can be a truncated protein as long as itretains its enzymatic activity to cause degreening (chlorophylldegradation). One skilled in the art would know that a truncated proteinmay be able to maintain enzyme activity (U.S. Pat. No. 4,762,914).Examples of chlorophyll degradation enzymes include, but are not limitedto, RCCR, PaO and chlorophyllase. The output/degreening circuit alsocomprises a target-substance-regulated inhibition of chlorophyllbiosynthesis. As specifically exemplified, this is achieved byexpression of either antisense, or preferably, interfering RNA molecule(such as diRNA, siRNA) sequences specific to a coding sequence for anenzyme in the chlorophyll biosynthetic pathway. These interfering RNAmolecules are examples of functional nucleic acids, and in the contextof inhibition of gene expression, a functional fragment of a codingsequence or gene is one which specifically interacts with a transcriptof the coding sequence or gene so as to reduce expression of the productof that gene or coding sequence. Examples of the enzymes involved inchlorophyll biosynthesis include, but are not limited to,protochlorophyllide oxidoreductase (POR), GUN4, other GUN genes (genomeuncoupling), Mg chelatase and chlorophyll synthetase. It is understoodthat other targets in the chlorophyll synthesis or degradation pathwaycan be substituted for those specifically set forth.

The DNA construct for transforming the readout or degreening genecircuit into a plant or plant cell typically contains a nucleic acidencoding at least one chlorophyll degradation enzyme (or a fragmentthereof which functions to effect chlorophyll degradation) and/ordesirably also a nucleic acid whose expression product inhibitschlorophyll synthesis operably linked to a promoter with transcriptionregulatory sequences that bind a transcription activator protein thatreceives the signal from the input gene circuit. Typically it can be atranscriptional activator protein that solely receives the signal fromthe transmembrane histidine kinase and shuttles to the nucleus or anuclear localized transcriptional activator protein that receives thesignal from the transmission protein which relays the signal from thetransmembrane histidine kinase and shuttles to the nucleus. The exteriorcomponent of the transmembrane histidine kinase has bound the sensorprotein substance complex therefore relaying an input signal generatedby an explosive, a chemical or biological agent, a pollutant or aspecific substance. In response to the input signal, this dualmodulation, i.e. inhibition of synthesis and stimulation of degradationof chlorophyll ensures loss of green color in plants when exposed to avariety of chemical agents or environmental pollutants. As describedherein, chlorophyll synthesis can be inhibited by producing interferingRNA or antisense RNA derived from at least one of the genes encodingchlorophyll synthetic enzymes.

Specifically exemplified herein is an output (degreening) circuitexpressing diRNA for POR and/or GUN4, and expressing proteins havingRCCR and/or PaO and chlorophyllase activities, all under the regulatorycontrol of a synthetic signal-receptive promoter (PlantPho) whoseexpression is controlled by an adaptive PhoB:VP64 signal receptive andtransaction protein and the input circuit comprising the sensor proteinwhich binds a target substance of interest. The transgenic plantscontaining the aforementioned degreening circuit lose green color, i.e.,turn white, within hours of exposure to the target substance. It ispossible to detect other changes in transgenic plants earlier in theprocess. A specifically exemplified input circuit comprises the SS-TNTsensor protein which interacts with one or more defined transmembraneproteins such as FLS:Trg:EnvZ:AHK4, or plant-bacteria hybridtransmembrane proteins such as FLS:Trg:PhoR or FLS:Trg:EnvZtransmembrane proteins (FIGS. 2A and 2B). The FLS:Trg:EnvZ:AHK4 proteintransmits a high energy phosphate signal to a plant histidinephosphotransferase protein specifically exemplified here, AHP, whichthen activates transcription. The plant-bacteria hybrid transmembraneproteins (FLS:trg:EnvZ:AHK4, FLS:Trg:PhoR and/or FLS:Trg:EnvZ proteins)transmit a high energy phosphate signal to an adapted protein,PhoB:VP64, with the result that the PhoB:VP64 transcription activatingprotein is phosphorylated, translocate from the cytoplasm to the plantnucleus and activate expression of the degreening genes operably linkedto the PlantPho promoter which is induced when the specific substance orsubstances are in the external environment of the plant or plant cell(FIG. 2C). This results in expression of the linked gene, in thespecific case of a plant sentinel the relatively rapid degreening, viathe inhibition of chlorophyll synthesis and the stimulation of thedegradation of chlorophyll, demonstrating that the DNA constructs of thepresent invention provide regulated gene expression of the detectablemarker.

Accordingly, a transgenic plant containing the input and output circuitsdisclosed herein loses its green color when exposed to a substance inthe environment which activates the input circuit by binding to aspecific receptor site (i.e., sensor protein) outside the plant. Thesubstance can be, for example, nerve gas, mercury, lead, arsenic,uranium, cadmium, selenium, polycyclic aromatic hydrocarbon, a benzene,a toluene, a xylene, or a halogenated (chloro, fluoro, and chlorofluoro)hydrocarbon, explosives, any substance listed on the EPA superfundwebsite, specific compounds involved in manufacture of compounds ofinterest, or a trigger substance to bring about a desired change in theplant or crop. In addition, the target substance which binds to aspecifically engineered sensor protein and input circuit via theextracellular receptor could be an explosive such as trinitrotoluene,RDX, HMX or TATP, or a degradation product of one of the foregoingcompounds specifically bound by the sensor protein.

The sensing and response system of this invention is modular in that itcan be coupled with a variety of input circuits (sensor proteins) toprovide specificity and selectivity for a particular chemical agentand/or other environmental factor of interest which is recognized by anavailable sensor protein that effectively interacts with the exteriordomain of the transmembrane protein when the target substance is bound.Similarly, the readout gene which is expressed via the histidine kinasesystem or systems of this invention can be selected for a desiredresult, with the proviso that it is operably linked to a promoter andassociated control sequences which interact positively with atranscription regulatory protein activated directly or indirectly by thehistidine kinase and/or AHP, PhoB or OmpR, described herein.Specifically, receptors which are engineered to bind site specific tothe target substance of interest (including but not limited to heavymetals, nerve agents such as soman or a degradation product thereof,such as pinacolyl methyl phosphonic acid), explosives and certaindegradation products thereof, environmental pollutants such as MTBE,herbicides such as glyphosate and the like. The sensing circuit furtherincludes the transmembrane protein with an external binding domain whichinteracts with the sensing protein-target substance complex and anintracellular portion which directs the phosphorylation of atranscriptional activator protein, as specifically exemplified by PhoBand/or modified and/or an adapted version of the PhoB protein. PhoB canalso be phosphorylated by an endogenous plant histidinephosphotransferase. The phosphorylated PhoB (activated form) then bindsto the PhoB cognate binding sequences which are part of the syntheticpromoter operably liked to a chlorophyll degradation enzyme codingsequence (such as chlorophyllase). The transcriptional activator proteincan also be a hybrid protein including but not limited to, PhoB:VP64translational fusion protein and it is expressed in a transgenic plantexpressing its coding sequence operably linked to a plant expressiblepromoter, which can be constitutive or which can include sequences fortissue-specific or condition-specific expression. The activator proteincan be any eukaryotic transcriptional activator including, but notlimited to VP16, VP64 and GAL4.

Histidine Kinase Signal Transduction System

Two component histidine kinase signal transduction systems are conservedbetween plants and bacteria (Stock et al., 2000; Koretke et al., 2000;Kakimoto, T., 2003; Kakimoto, T., 1996) and this conservation was thebasis of forming a functional input (sensing) circuit.

In bacteria, sensitive chemotactic sensors exist to direct motilebacteria to nutrients, e.g., ribose. When a periplasmic binding proteinsuch as the ribose binding protein binds its ligand, it develops a highaffinity for the extracellular domain of bacterial chemotactic receptorssuch as Trg. Upon binding of the ligand/binding protein complex, acytoplasmic histidine kinase is activated. Normally in the bacterium,this results in chemotaxis toward the food source. Hybrid histidinekinases have been expressed in bacteria where the cell surface PBPbinding domain of Trg has been combined with the interior histidinekinase domain from proteins such as envelope Z (EnvZ) (Looger et al.,2003; Baumgartner et al, 1994). This hybrid protein activatestranscription via phosphorylated transcription activator proteins. Inthe hybrid histidine kinases, the target substance is bound by thesensor protein, and the substance:protein complex binds to theinteracting domain of the hybrid histidine kinase at the exterior sideof the cell membrane, and that initiates activation of the histidinekinase (HK). The HK starts a phospho-relay (phosphorylation relay)through a bacteria response regulator (e.g., OmpR or PhoB) to activatetranscription of bacterial genes. The phospho-relay always goesHis→Asp→His, etc. In addition, at least some transcription activatorproteins are phosphorylated (activated) by that same kinase domain.

Chemotactic binding proteins (periplasmic binding proteins) have beenredesigned using computer-run computational design methods so thatinstead of binding substances such as ribose or galactose or maltose,the engineered proteins specifically bind a target substance of interestsuch as TNT, RDX, nerve gas, heavy metals, or other environmentalpollutants or harmful substances.

Plants also use a two-component or histidine kinase signaling systemthat responds to cytokinin (a plant hormone). Plant signal transductionis more complex. The histidine kinases are “hybrid types”. The plant HKsin Arabidopsis are known as AHKs. Upon sensing cytokinin, plant HKsphosphorylate an internal histidine kinase and initiate a phospho-relayinternally to an aspartate residue located in the receiver domain of thesame protein. The receiver domain transfers the phosphate group to anindependent protein (AHP, Arabidopsis histidine phosphotransferase). TheAHP moves into the plant cell nucleus upon phosphorylation and thentransfers the phosphate group to a nuclear localized protein, ARR TypeB, transcription factors that then initiate transcription of ARR Type Agenes. Examples of ARR type A genes useful in the present inventioninclude, but are not limited to, ARR5 and ARR7, or any Type A ARR gene.Other functionally equivalent sequences may also be used in the systemsdescribed herein.

Computer design enables the design of sensor proteins to bind with greatspecificity and sensitivity, a variety of compounds or substances. See,for example, US Patent Publications 2004/0118681, and 2004/0229290;Looger et al. (2003); Dwyer et al., (2003) and Allert et al. (2004).Periplasmic binding proteins as starting points for protein engineeringare reviewed in Dwyer et al., 2004. In bacteria, the engineeredreceptors were targeted to the periplasmic space to sense varioussubstances of interest. In plant cells, it is necessary to add(desirably at the N-terminus) a secretory sequence functional in plantcells so that the sensor protein is at the exterior of the cell and canbind the particular target substance of interest and it is necessary todelete the bacterial leader. The starting point is the engineeredperiplasmic binding protein, and the ending point is a detectable changeresulting from a transcriptional response in the nucleus;computer-designed sensor proteins and molecular biological techniquesallows for the combination. Hybrids at both the starting point andending point allowed functional signaling.

To obtain information from outside the plant cell and transmit a signalto the nucleus of the plant cell, specifically engineered target sensingreceptors were positioned outside of plant cells. The proof of conceptwork was done with the receptor or sensor protein for TNT(trinitrotoluene). The original receptors that are computationallydesigned are the periplasmic binding proteins: RBP, MBP (maltose bindingprotein) and GBP (galactose binding protein). Importantly, at least inpart because the system is modular, PCR can be used to change thereceptor/sensor protein portion from a receptor/sensor protein specificfor TNT to a target substance of interest (RDX, nerve gas, zinc, heavymetal, environmental pollutant).

Plant Extracellular Space:

Plants are not known to have a functional periplasmic space. However,evidence indicates that there is a functional space between the plantplasma membrane and the outside. Small proteins can freely move and/ordiffuse in the plant cell wall, better understood as a complex matrix,and even move and/or diffuse in the plant cuticle, the waxy coating thatis found outside some plant organs (Baluska, F et al., 2003; Somerville,C. et al., 2004). In bacteria, the periplasmic binding protein containsa leader peptide portion that targets the protein to the periplasm. Inplants, proteins are targeted to the extra-cellular space by way of theendoplasmic reticulum. Because of the different targeting mechanisms, aplant extracellular targeting sequence is needed and the bacterialperiplasmic targeting leader must be removed.

Genetically Engineered Plants Capable of Losing Green Color:

The present invention also provides genetically engineered plantscapable of losing green color in response to a signal (analyte orligand) by simultaneously controlling expression of genes involved inchlorophyll biosynthesis and/or degradation. These plants are capable ofreceiving input from cytoplasmic and extracellular analytes and linkingthese components to the degreening circuit resulting in the loss ofgreen color. Thus, the plants of this invention serve as a simple andeasily detectable biomarker for adverse environmental input.

The degreening circuit is assembled in a “plug and play” manner. Hence,the sensor protein for TNT, which initiates the input, can be replacedby a different computationally designed sensor protein allowing thedegreening circuit to respond to a specific target substance or targetsubstances of interest. The principle of the invention is illustratedusing the model plant species Arabidopsis, which allows rapidoptimization of the degreening circuit and its response. However, thecircuits described herein are readily introduced into other plantspecies such as those typical of shopping malls, office buildings,landscapes, forested areas, cropland or aquatic systems.

The plants of this invention that lose their green color in response toa target substance can serve as untiring sentinels reporting on adverseinput from the environment (e.g., chemical weapons or pollutants). Plantsentinels would be unthreatening to the general public and can bedeployed in shopping malls and office buildings and at special eventswhere most people can recognize a loss of green color and securitypersonnel could easily detect the changes within a short period withinexpensive hand-held machines. In addition, loss of green color orother disruption of chlorophyll, such as chlorophyll fluorescence, orphotosystem electron transport or hyper-spectral imaging can be rapidlyquantified by authorities with either portable hand-held equipment orsimple laboratory equipment (spectrophotometers). In vast geographicareas, detector systems could be introduced into plants typical forlandscapes and aquatic systems, allowing satellites to identify adverseenvironments.

The degreening circuit of the invention induces genes that are involvedin chlorophyll breakdown and synthetic genes for inhibiting chlorophyllsynthesis. Simultaneous expression of the genes that initiatechlorophyll breakdown and inhibit new chlorophyll biosynthesis wouldyield the most efficient degreening phenotype. For this reason, thedegreening circuit exemplified herein was created using three genes, twoin the chlorophyll degradation pathway and one inhibitory gene in thechlorophyll biosynthesis pathway. A person of ordinary skill in the artunderstands that other combinations of the genes that are known to beinvolved in chlorophyll synthesis and degradation can be used to obtainthe degreening phenotype demonstrated herein. In addition, a person ofordinary skill in the art understands that the reactive oxygen species(ROS) generated in the chloroplast and reported in Antunes et al., 2006,could be used to initiate and generate the degreening within plastids.

The degreening circuit of the invention can respond in two differentways; it can respond to target substances within the cytoplasm as wellas those that are extracellular. To test the ability of the degreeningcircuit to function with cytoplasmic input in plants, a syntheticcytoplasmic receptor is linked to the circuit. In response to binding ananalyte, the cytoplasmic receptor is transported to the nucleus where itactivates synthetic transcriptional promoter(s) fused to genes whoseproducts degrade chlorophyll while preventing new chlorophyllbiosynthesis. To test the ability of the degreening circuit to functionwith input from outside the plant, an input circuit containing achimeric receptor or binding protein can be linked to the degreeningcircuit. In response to binding an analyte, the extracellular receptorinitiates a signal transduction pathway and activates a signal receptivesynthetic transcriptional promoter fused to genes whose products degradechlorophyll while preventing new chlorophyll biosynthesis.

Normal time periods for notable loss of green color in plants varieswidely from days to weeks depending on whether the loss is triggeredfrom environmental changes, development (e.g., flower petals) or stress(e.g., pathogens). To develop a system that can lose green color rapidlyin response to a signal, both the chlorophyll biosynthesis andchlorophyll breakdown pathways were modified to construct a “degreeningcircuit”. The degreening circuit is shown schematically in FIG. 1F. Inaddition to genes involved in chlorophyll metabolism, a redundantmarker, green fluorescent protein (GFP) can be included in thedegreening circuit as a control. The GFP marker is similarly(optionally) linked to the input part of the circuit and serves toeliminate false positives that might arise.

There has been only one study where genes involved in chlorophyllmetabolism have been purposely altered in transgenic plants (Benedettiand Arruda, 2002). In their study, the chlorophyllase gene wasover-expressed in Arabidopsis plants and the plants remained green, buthad an enormous increase in the level of the breakdown productchlorophyllide. To ensure that the degreening phenotype appears rapidly,two genes (for example, chlorophyllase and RCCR) were used in thedegreening circuit exemplified herein. Although it was not measured, theturnover in chlorophyll is strongly believed to have stimulated feedbackinduction of new chlorophyll biosynthesis. To prevent this fromoccurring in the degreening circuit, expression of theprotochlorophyllide oxidoreductase (POR) gene, the rate-limiting enzymein chlorophyll biosynthesis was inhibited.

One approach to prevent expression of (silence) a specific gene involvesthe production of an interfering RNA molecule that contains a sequenceidentical to the gene of interest (McManus and Sharp, 2002; Wang andWaterhouse, 2002). Typically, the plants are genetically engineered toexpress inverted repeats (500-700 bp) to the gene of interest. Theresulting double-stranded RNA is homologous to an endogenous transcript.Transgenic plants containing diRNA show high turnover rates of thehomologous transcript and complete silencing of the endogenous geneexpression (Chuang and Meyerowitz, 2000; Welsey et al., 2001; Wang andWaterhouse, 2002). An interfering RNA molecule has been shown to be moreefficient than antisense RNA in blocking the expression of a desiredgene with silencing frequency between 90-100% (Waterhouse et al., 1999;Chuang and Meyerowitz, 2000; Smith et al., 2000; Welsey et al., 2001;Stoutjesdijk et al., 2002; Wang and Waterhouse, 2002). Based on thesestudies, the initial degreening circuit is generated using doublestranded RNAs to silence the POR gene in a transgenic plant and henceprevent the de novo synthesis of chlorophyll after input from ananalyte. A series of convenient Arabidopsis vectors for making dsRNAconstructs (developed by Jorgensen and Chandler) can be obtained fromthe Arabidopsis Biological Resource Center, Ohio State University. Thesevectors are based on the binary vector pBCAMBIA1200 (Cambia, BlackMountain, AU) and contain a cassette for cloning a desired gene or geneportion in the sense and antisense orientations. The cassette has twopairs of unique restriction enzyme recognition sites flanking a 335 basepair GUS (β-glucuronidase) fragment that separates sense and antisenseregions of the inverted repeat and facilitates formation of the dsRNA.The vectors are a series of plasmids that replicate in both E. coli andAgrobacterium tumefaciens allowing easy cloning and planttransformation, respectively. Vectors are available carrying the Bar orNptII genes, the plants containing the introduced genes can be selectedwith the herbicide BASTA (glufosinate ammonium) or the antibiotickanamycin, respectively. A chloamphenicol or spectinomycin gene providesbacterial selection. The conserved region of protochlorophyllideoxidoreductase (POR) gene is cloned as described below in the sense andantisense direction to produce the diRNA molecule specific for the PORgenes. The vectors are designed to direct expression of the diRNAmolecule with a strong constitutive promoter (CaMV 35S). To place thediRNA vector in the degreening circuit, this promoter, which is flankedwith unique restriction sites, is replaced with promoters that placeexpression under control of perception of cytoplasmic or extracellularanalytes for example, using the Pho promoter described.

Plant transformation methods are routine, with Arabidopsistransformation among the easiest. Arabidopsis transformation is easilyaccomplished by spraying, dipping or contacting flowers with a solutionof disarmed Agrobacterium tumefaciens containing the genes of interest(Bechtold et al., 1993; Clough and Bent, 1998; Chung et al., 2000;Desfeux et al., 2000). The various constructs can all be introduced intobinary plant transformation vectors that provide the plants withresistance to kanamycin, BASTA or hygromycin. Once the individualcomponents are assembled in binary plant transformation plasmids, theyare then introduced into a disarmed strain of Agrobacterium tumefaciens(e.g., ASE or GV4111)(Fraley et al., 1985). The Agrobacterium cells arethen grown to an A₆₀₀ of 0.5 in one liter batches, prepared as described(Clough and Bent, 1998; Chung et al., 2000) and effectively contactedwith flowering Arabidopsis plants. The plants are allowed to set seed,the mature seed collected and transformed plants selected by eitherresistance to the antibiotics kanamycin and hygromycin or herbicideresistance to BASTA.

Assembly and Testing of Degreening Gene Circuits.

In many biological responses, sensing of a specific substance leads to atranscriptional response. The synthetic sensing system for plantsentinels links input to transcriptional output (Looger et al., 2003);hence, we created a test readout system triggered by a transcriptionalresponse (signal-regulated induction of gene expression). Numeroustranscriptional induction systems are available which provide a model inwhich to test the chlorophyll reporter system. A synthetically designed,steroid inducible system was modified to function in plants. In thepresence of a synthetic steroid (4-hydroxytamoxifen, 4-OHT), a chimerictranscriptional regulator relocates to the nucleus and inducesexpression of a promoter made up of specific response elements and the−46 region of the CaMV35S promoter, designated 10XN1P. The 4-OHTinduction system is essentially analogous to other transcriptionalinducible systems (Zuo et al., 2000).

In order to use plants to monitor large areas for pollution or terroristagents, a reporter or readout system is needed. Prior gene reportersystems were developed for laboratory use and do not providecharacteristics needed for a plant sentinel. A synthetic degreeningcircuit was developed that allows the green pigment chlorophyll to beused as a biosensor readout system. Induction of the degreening circuitallows remote detection, displays a rapid response, provides a resetcapacity, and results in a phenotype readily recognized by the generalpublic. Because the degreening circuit produces a white phenotype, it iseasy to distinguish it from plants stressed from biotic or abioticconditions, which produce yellow (or other color) phenotypes viasenescence-related pathways. The inability to reset biosensors has beenthe major limitation to their use. The degreening circuit provides asimple capacity to be reset. Plants regreened after removal of theinducer, and this regreening was enhanced by a brief cytokinintreatment. Because the transcriptional inducer used (4-OHT) isrelatively stable, the degreening circuit may not fully switch to an“off” position immediately following removal of the inducer, and theregreening process may not start until the inducer within the plantdegrades. Hence, it should be possible to substantially reduce the timeneeded for regreening, currently 3 days.

It should also be possible to reduce the response time from less thantwo hours to minutes. Our initial time point at two hours detectedsubstantial reduction in φ_(PSII), one of the most robust parameters inchlorophyll fluorescence imaging. The rapid decline seen at two hourssuggests we could detect changes earlier; indeed, detailed statisticalanalysis of the fluorescence parameters should allow us to accuratelydetermine when the first significant changes can be detected. Inaddition to improving remote detection ability, the genetic circuitrycould be further enhanced by rationally applying principles developedfor synthetic gene circuitry (McDaniel and Weiss, 2005). For example,gene circuitry could be designed to be activated and remain active upona single exposure to a small amount of inducer (via the sensing pathway)using a trigger function as described in FIG. 9.

The degreening circuit, combining “stop-synthesis” with an “initiatebreakdown” function, caused loss of chlorophyll with unprecedentedspeed. When each function was introduced separately, plants did notvisibly degreen in the 48 hour timeframe except in the cotyledons.Expression of the “initiate degradation” circuits (CHLASE and PAO, orCHLASE and RCCR) failed to produce rapid degreening, suggesting thatplants can enhance chlorophyll biosynthesis when needed. Likewise, the“stop synthesis” circuits (diRNA specific to POR or GUN4) failed toproduce rapid degreening, supporting the concept of a large amount ofmetabolically stable chlorophyll within the plant. The rationalcombination of these two functions in one T-DNA construct produced asynthetic “degreening circuit”. The designed gene circuit is successfulwith respect to signal responsiveness, as indicated by three types ofdata: response of excised leaves to dark-induced senescence, distinctiveultrastructural changes, and microarray data showing a difference ingenes regulated by the degreening circuit and normal chlorophyll loss insenescence.

Because of the massive damage to the photosystems, evidence of reactiveoxygen species (ROS) was determined. FIG. 5D shows massive accumulationof ROS in the degreening leaf cell but not in the untreated leaf cell.

Light was shown to be important for the rapid degreening process tooccur, as induced plants incubated in the dark failed to turn white,even after 72 hours of induction. When induced plants were transferredto light, degreening proceeded at an enhanced rate (FIG. 5E). Theseresults suggest that the degreening circuit is poised to respond indarkness, but not able to initiate rapid degreening without light.Chlorophyll biosynthesis and breakdown intermediates are potentiallyphototoxic (Matile et al., 1999). Because the degreening circuitinterferes with the normal balance of chlorophyll and likely itsmetabolic intermediates, it is possible that, upon light exposure, thesemolecules cause photo-oxidation of pigments. A similar light requirementfor degreening was observed for detached leaves. Under standard lightconditions degreening induction caused detached leaves to fully degreenwithin 48 hours. However, darkness failed to induce full degreening indetached leaves, even after 72 hours of induction. Because darkness hasbeen shown to induce senescence in Arabidopsis detached leaves (Weaverand Amasino, 2001), these results suggest that chlorophyll loss from thedegreening circuit is distinct from senescence.

Because light is required for rapid chlorophyll loss, we looked at howlight is handled by plants induced to degreen by the detection of anenvironmental signal. The use of remote measurements of chlorophyllfluorescence provides an easy detection system for plant sentinels.φ_(PSII) measures the proportion of the light absorbed by chlorophyllassociated with photosystem II (PSII) that is used in photochemistry. Itis an indication of the plant's overall photosynthesis (Maxwell andJohnson, 2000). Degreening plants show a rapid decline in φ_(PSII)within 2 hours of induction, suggesting that induction of the degreeningcircuit quickly disrupts photosynthesis, and provides a quick way ofdetecting changes in plant sentinels. F_(v)/F_(m), a widely-usedparameter used for assessing the plant's level of stress, has an initialvalue of 0.8 in uninduced plants, indicating non-induced plants are notstressed. Induction of the degreening circuit causes a decline inF_(v)/F_(m) values, when compared to controls, also an indication thatthe photosynthetic ability of degreening plants is disrupted.

Decreases in F_(v)/F_(m) typically result from a combination of twoprocesses: increases in the rate constant for thermal dissipation and/ordecreases in the rate constant for photochemistry. Because light isrequired and the plants lose chlorophyll and yellow pigments, onepossibility is that excitation energy is dissipated from chlorophyll byinteraction with xanthophylls and other accessory pigments.De-excitation of chlorophyll was shown to occur by a rapidly reversibleelectron exchange between chlorophyll and zeaxanthin (Holt et al.,2005). If chlorophyll dissipates energy through zeaxanthin, we predicteda substantial change in non-photochemical quenching (NPQ). However,while NPQ measurements changed with induction, these changes werevariable both in time and among the degreening circuits, indicating thatit was not the primary means through which the degreening circuitfunctions. Hence, the decrease seen in F_(v)/F_(m) was not primarilycaused by enhanced thermal dissipation. Because φ_(PSII) and F_(v)/F_(m)decrease prior to substantial decreases in chlorophyll levels, our datasuggested that the degreening circuit functions by the inactivation orremoval of PSII cores, which precedes substantial removal ofchlorophyll. If the degreening circuit functions through inactivation orremoval of PSII cores, there should be a large production of reactiveoxygen species (ROS).

In degreening plants, ROS was first detected after 8 hours withsubstantial accumulation of ROS seen at 30 hours, consistent with thehypothesis that the degreening circuit functions by loss or damage ofPSII cores. A mechanism proceeding via ROS action on photosystem coreswould also account for the results that degreening circuits with varyinggene compositions all produce a similar phenotype.

If ROS and/or damage to photosystem cores are key to initial degreeningcircuit function, the microarray analysis at 24 hours should indicatethat genes typically involved in photosystem repair are significantlydown-regulated. DegP2, encoding a protease that is responsible forinitial repair of damaged PSII proteins (Haussuhl et al., 2001) isdown-regulated. In addition, FtsH6, a chloroplast LHCII protease(Zelisko et al., 2005) is likewise down-regulated. Further analysis ofmicroarray data suggests that various PSII- and PSI-related genes aredown-regulated, while ROS-related genes are simultaneously up-regulated,indicating a process largely distinct from normal chlorophyll loss insenescence.

The degreening circuit provides an effective means to controlchlorophyll levels in plants. The trigger for the degreening circuit isa specific input, resulting from sensing of the binding of a targetsubstance of interest outside the plant, with signal transduction viahistidine kinase within the cell and nuclear transcription activation.The steroid-inducible 10XN1P promoter used with the degreening circuitas a model can be replaced with other promoter elements, such as thoseresponsive to signal transduction (Sakai et al., 2000) or the syntheticPlantPho promoter, as readily understood in the art. By combining thecontrolled chlorophyll loss as a reporting element with a sensing systemsuch as computationally designed receptors or sensor proteins thatprovide input via transmembrane histidine kinases (Allert et al., 2004;Looger et al., 2003), plants are produced to serve as inexpensivemonitors for terrorist agents, environmental pollutants or other targetsubstances of interest. Degreening indicating presence of the targetsubstance can be observed visually at close range or detected from adistance by remote sensing, as known to the art.

All DNA constructs, transgenic plant cells, tissue and plants, andmethods for detecting a target substance of interest or for obtaininggene expression in response to the presence of the target substance ofinterest are within the scope of the present invention. It is furtherunderstood that other evolutionarily conserved signal transductioncomponents and systems, transcription regulatory components and can besubstituted for those recited herein, provided that there are functionalinput and/or output circuits responsive to the presence of a targetsubstance.

EXAMPLES

The following examples are provided for illustrative purposes, and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified articles which occur to the skilledartisan are intended to fall within the scope of the present invention.

In plant pollination, a large number of proteins are targeted to theextracellular space as a means of protein pollen-pistil (male-female)recognition. An example of one of these proteins is PEX (PollenExtension-like protein). The leader peptide from a pollen protein calledPEX (At1g49490=PEX) targeted proteins outside the pollen during thefertilization process (Baumberger, N. et al., 2003). Using overlappingPCR technology the coding sequence for this leader was fused to theperiplasmic binding proteins' coding sequences to target them to theextra-cellular space in plant cells. In addition, an N-terminalbacterial leader which properly targets the periplasmic binding proteinto the periplasm in bacteria was removed (interfered in proper targetingin plant cells). The PEX SS(SS, secretory sequence) and thecomputationally designed RBP coding sequence yielded a chimeric codingsequence of 900 bp (SS:TNT). See SEQ ID NO:7 and SEQ ID NO:8.

Fusion proteins with GFP (green fluorescent protein) and the abovelisted constructs were made and transient assays (particle bombardmentof nucleic acid constructs into onion epidermal cells) were used todetermine if any of these constructs were properly targeted to theoutside of the plant cell. These assays showed that SS:RBP was presentthroughout the cell, with no specific localization to the plant cellwall. Removal of the bacterial leader and addition of the PEX leader wasfound to properly target the periplasmic binding protein to theextracellular space, the cell wall matrix.

It is possible that the highly hydrophobic nature of the nativebacterial signal peptide might interfere with the plant signal peptideand interfere with proper targeting. In the ssRGDRBP fusion protein, thePEX SS is separated from the RBP hydrophobic signal peptide by the RGDmotif. This separation kept the bacterial sequence from interfering withthe PEX SS. Two new ssRBP fusions were also designed. The first fusionhas the RGD motif minus the amino acids RGD between the SS and RBP(ss-linker-RBP), and the second fusion had the SS fused to RBP with thebacterial hydrophobic signal peptide deleted from the RBP protein (ssRBPno RBPss). Both fusions were able to target the RBP outside of theplasma membrane.

Constructs described above properly targeted the periplasmic bindingprotein to the plant cell wall. The bacterial hydrophobic leader wasremoved and replaced in its entirety with the plant leader (SS PEX). Theamino acids RGD were removed from the linker and the linker was used toseparate the plant leader (SS-PEX) from the bacterial leader.

Cells were plasmolysed to show proper targeting of the periplasmicprotein (in this case RBP) to the plant extra-cellular space.Plasmolysis causes the plant protoplast to shrink away from the cellwall revealing localization of the periplasmic protein in theextracellular space. Plasmolysis of cells expressing the GFP fusionprotein showed that targeting takes the periplasmic protein to theextra-cellular space. The data suggested that the targeted periplasmicbinding protein fusion (SS-RBP:GFP) can freely diffuse in the cell wall.

A quick assay for reliable function was developed to show propertargeting of bacterial histidine kinases in plants. Plant systemstransfer two-component signaling information from the membrane localizedhistidine kinase to the nucleus via histidine phospho-transferases. Inplants, all published reports indicate this transfer is transient. Inbacteria, information is transferred from the membrane localizedhistidine kinase to the bacteria chromosome via proteins called responseregulators.

In planta nuclear shuttling ability of both plant AHPs (histidinephosphotransferases) and bacterial response regulators usingtranslational GFP fusions were tested. The fusions were first tested intransient assays and then stably introduced into transgenic plants. Inbacteria, both the plant AHPs and bacterial RRs function in a signaldependent manner. Hence, it is possible to test both the normal plantAHPs and the bacterial RRs using a normal plant signaling molecule, thehormone cytokinin, using evolutionary functional cross-talk betweenkingdoms.

Bacterial Response Regulators:

Bacterial response regulators and other heterologous signalingcomponents are determined to be able to function in plants and shuttleto the nucleus. Two bacterial response regulators (PhoB and OmpR) werefused to GFP and transformed into Arabidopsis plants. OmpR is thebacterial response regulator which has been used in certain engineeredsystems. In bacteria, OmpR receives a phospho-relay signal from thebacterial histidine kinase EnvZ (envelope Z), with the result thataffinity for bacterial promoter elements is increased. PhoB is abacterial response regulator that will also pick up a phosphorylationsignal from EnvZ, although its natural histidine kinase is the bacterialPhoR. Once PhoB receives a phosphorylation signal, its conformationchanges and it develops a high affinity for bacterial promoters. Whenthe phosphorylated PhoB protein is bound to the promoter, transcriptionis activated. In plants this was studied with the GFP fusion in leaves,petioles, roots and the crown (stem-like region in Arabidopsis). FIGS.3A and 3B show that a signal-(cytokinin) dependent movement of thebacterial response regulators into plant cell nucleus was observed.There was a signal-dependent movement of the bacterial responseregulators into the plant nucleus in every tissue and cell typeexamined. The PhoB:GFP shows strong signal dependent nuclear shuttling.OmpR also showed signal dependent nuclear localization, through somewhatless efficiently.

Transgenic roots were examined using a confocal microscope to confirmthat the bacterial response regulators (PhoB:GFP, OmpR:GFP) moved intothe nucleus, rather than associating with the nuclear membrane. If thePhoB and OmpR fusions were caught at the nuclear membrane, a roundish or‘donut’ shape would be seen in a confocal microscope. However, confocalmicroscopy of PhoB:GFP transgenic roots indicated that there was nuclearlocalization in response to the cytokinin signal. There was no evidenceof PhoB at the nuclear membrane. OmpR:GFP also is localized to thenucleus, but showed less shuttling than the PhoB:GFP fusions. Thus, thebacterial response regulators (PhoB and OmpR) act as membrane-to-nucleusshuttling proteins for the phospho-relay.

To rule out the possibility that the bacterial response regulatorsaccumulate in the nucleus simply because they can diffuse through thenuclear membrane (both proteins have a molecular mass of approximately27 kDa), a larger fusion protein was made by adding the β-glucuronidase(GUS) coding region to the C-terminal end of the PhoB:GFP fusionprotein. The resulting protein, PhoB:GFP:GUS, has a predicted molecularmasses of 122 kDa. This larger fusion protein was transformed intoArabidopsis and transgenic plants analyzed. Transgenic roots wereexamined before and after the cytokinin treatment under anepi-fluorescence microscope. FIG. 3C shows that PhoB:GFP:GUS fusionprotein accumulates in the nucleus after cytokinin treatment. DAPIstaining confirmed the compartments' identity as nuclei. Therefore, thebacterial response regulator PhoB move into plant nuclei in asignal-dependent manner, and that the movement is not through diffusion.

In bacterial cells, PhoB and OmpR proteins are phosphorylated atconserved Asp residues (D53 in PhoB and D55 in OmpR) by their cognatehistidine kinase. Transgenic plants were generated that containedmutated forms of PhoB:GFP and OmpR:GFP, where the conserved Asp residueswere mutated to Ala. FIG. 3D shows that fluorescence fromPhoB^(D53A):GFP is diffuse in an untreated root. The same root aftercytokinin treatment shows a different pattern of PhoB^(D53A):GFPlocalization when compared to wild-type PhoB:GFP. A detailed view of theroot shows that nuclear localization of PhoB^(D53A):GFP is variable andsporadic (arrowheads point to nuclei). In most cases, PhoB^(D53A):GFPseems to accumulate at the base of cortical cells (arrows and somenuclear accumulation was seen in vascular cells only. In contrast,mutation of Asp55 in OmpR completely abolishes its signal-dependentnuclear shuttling.

Hybrid Histidine Kinases:

Because bacterial cell membranes and plant cell membranes differsubstantially in components, it had not been expected that both theextracellular receptor which responds to the computationally designedperiplasmic binding proteins and the transmembrane histidine kinasecould be derived from bacteria. It was believed that hybrid histidinekinases with plant and bacterial segments would be required for thetranscription regulatory circuits of the present invention to befunctional. Certain hybrid histidine kinases had already been made withbacterial components. However, with the incorporation of a plantsecretory sequence, both types of histidine kinases were successful.

While the specifically exemplified transmembrane proteins describedherein include segments from the bacterial histidine kinase TRG (or TRZ)and the plant AHK4 histidine kinase, other transmembrane proteins cansupply the kinase function in a chimeric transmembrane protein.Additional histidine kinases are described in Inouye, Mand Dutta, R.,eds. (2003) Histidine Kinases in Signal Transduction, Academic Press,NY. Examples of bacterial HKs which can be utilized in the transmembraneprotein of the present invention include por S, arc B, bar A and evg S.A Trz-PhoR fusion with a plant signal sequence has also been produced;see SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:12.

It was discovered that in building up complex phosphate relays usingprotein domains with strong functional homology from different organisms(yeast, bacteria, and plants), the best fusion points for preservingfunctionality are unexpectedly within the domains at homologoussequences that are involved in the actual function of the domain. Anexample of such a fusion is the TRZ-AHK4 fusion, which has been adaptedfor plant gene expression and targeted to the cell membrane. The fusionis within the G2 (GLGL/I) box in the hATPase domain of the histidinekinase. Fusions at the end of the domains did not function properly.

One of the bacterial histidine kinases that is capable ofphosphorylating PhoB and OmpR is TAZ. TAZ is a previously characterizedhybrid of two types of bacterial kinases. It has an aspartate receptorfrom the chemotactic receptor Tar in the extracellular space fused tothe EnvZ component inside the membrane. The gene fusion is at an Ndesite at the end of the Tar HAMP domain. TAZ sends a signal dependentphospho-relay to PhoB, and PhoB (and PhoB:GFP fusion) upon receiving asignal, shuttled to the nucleus in a signal-dependent manner. TAZfunction in plant cells using nuclear shuffling of PhoB:GFP was used asan assay. It is known that plant histidine kinases are known to functionin bacteria and hence as shown herein, bacterial histidine kinases canfunction in plants and that the transmembrane activation mechanisms areconserved.

Bacteria-Plant Hybrid:

One way for getting the computationally designed receptors to work inplants is forming a bacteria-plant hybrid molecule at some point in thesignal transduction pathway. One powerful aspect of the two-componentsystems used here is that they provide a molecular foundry for quicklytesting constructs' function in bacteria and then transferring thefunctional components into plants (with the appropriate plant leadersand expression components added). It has been shown that plant histidinekinases could activate bacterial signal transduction pathways causingthe signal-dependent production of the reporter gene β-gal. Multiplefusions of bacterial and plant histidine kinases were studied. Aidingthis process was a modular foundry where the fusion could be rapidlytested in bacteria and a plant leader added and the same fusion testedin plants.

The bacterial histidine kinase TRZ, a known functional hybrid betweenTRG (which binds the periplasmic binding protein RBP) and the histidinekinase EnvZ (which signals to the response regulator OmpR), was used toform a fusion with the plant histidine kinase AHK4 (At2g01830). Inplants, AHK4 signals via a phospho-relay to the plant AHPs. Thephosphorylated plant AHP moves into the nucleus and activates expressionof nuclear localized transcription factors, called type B ARRs. Type BARRs activate transcription of cytokinin responsive genes including thetype A ARRs. One of the ARRs that AHK4 activates is ARR5. Plantscontaining the ARR5 promoter fused to the plant reporter genes GFP andGUS were described in Romanov et al. (2002). In addition, the ARR7promoter can also be used to regulate GFP expression in transgenic plantcells. However, the promoter for any type A ARR would also work.

The functional bacteria:plant HK was produced by a fusion at the G2domain of the histidine kinase ATPase. The G2 domain is common to bothTRZ and AHK4. The fusion essentially adds the receiver domain of AHK4 tothe bacterial histidine kinase, in effect, synthetically evolving thebacterial histidine kinase ‘upward’ to a plant histidine kinase (FIG.2). The bacteria:plant hybrid histidine kinase was found functional inbacteria. To determine if the bacteria:plant hybrid histidine alsofunctioned in plants GUS/GFP expression was monitored from either theARR5 or ARR7 promoters. Hybrid histidine kinases found to be functionalin bacteria were modified for proper expression in plants using aplasmid construct containing the NOS promoter:plant leader(FLS):hybrid-HK:NOS terminator (NOS 3′). In addition, the test plasmidalso contained a construct called ssTNT. See SEQ ID NO:7. This is thesecretory construct shown to target periplasmic binding proteins tooutside the plant when fused to the designed TNT receptor.

A “synthetic” signal transduction pathway was constructed. It wasdetermined that the bacterial response regulators (PhoB, and to a lesserextent OmpR) moved into a plant nucleus in a signal dependent manner.PhoB was the response regulator of choice because of its strongsignal-dependent nuclear localization and because it is wellcharacterized in bacterial systems.

Bacteria, yeast, and plants can sense aspects of their environmentthrough conserved, two-component or histidine kinase (HK) signaltransduction systems. The protein components of these systems aretypically comprised of multiple, relatively modular domains, arranged invarious combinations and compositions. These domains tend to beconserved across pathways and species. Transfer of phosphates betweencomponents can exhibit a considerable amount of cross-talk, establishingnetworks that integrate multiple signals, rather than linear pathwaysthat link a single stimulus to a response. Sequence conservation andcross-talk was determined to be extended across kingdoms and thereforeused to establish a synthetic signal transduction system in a plant. Thecross-talk between HK systems was utilized and the bacterial responseregulators, PhoB and OmpR, were adapted for plant function. In responseto cytokinin-mediated HK signaling, these bacterial proteins shuttled tothe plant nucleus.

PhoB shuttling to the plant nucleus is not simple diffusion; some typeof active transport is required. Nuclear pores in plant cells excludemolecules larger than 60 KD. One possibility for the observedsignal-dependent shuttling of PhoB is that it is simply an increasedaccumulation in the nucleus. For example, PhoB is a small protein andcould freely diffuse in and out of the nucleus. Upon phosphorylation,PhoB's affinity for DNA could cause it to non-specifically bind a plantDNA. To test this, a PhoB:GFP:GUS fusion where the predicted protein is120 KD was produced. This allowed for testing whether PhoB freelydiffuses or whether it is transported. A GUS reporter (β-glucuronidase)was fused at the C-terminus of PhoB:GFP. The protein fusion constructswere transformed into Arabidopsis plants, always selecting plants with asingle T-DNA insert and then movement of the PhoB:GFP:GUS fusion proteininto the nucleus in a signal-dependent manner was monitored. Prior tocytokinin treatment, PhoB:GFP:GUS was found diffused throughout thecells with some weak accumulation in the nuclei. The weak nuclearaccumulation may be due to endogenous histidine kinase signaling coupledwith the inability of the PHoB:GFP:GUS protein to leave the nucleus.However, after cytokinin treatment PhoB:GFP:GUS nuclear accumulation wassignificantly enhanced. These data indicate that the nucleartranslocation of PhoB and OmpR cannot be explained by diffusion,indicating that they are transported into the nucleus and that plantfactor(s) unexpectedly recognize conserved regions of these bacterialresponse regulators.

Cytokinin induced PhoB movement to the nucleus does require aspartateused in bacterial signaling. Two component signaling pathways involveHis-Asp phosphor-relays. In bacteria, the PhoB response regulator isnormally phosphorylated at an Asp residue (D53). It was then determinedif this critical Asp is also used in planta. In plants, transmembranehistidine kinases autophosphorylate at a His residue and then transferthe phosphate to an Asp residue within the protein. Normally, thephosphate is then transferred to a His on the histidinephosphotransferase proteins, AHPs.

The nuclear shuttling unexpectedly requires the bacterial conservedphosphor-accepting Asp residue. Because of this discovery, PhoB's DNAbiding and transactivation properties were used and adapted to functionin plants, in combination with a synthetic, PhoB-responsive, promoter(PlantPho). In response to a cytokinin signal the adaptive protein,PhoB:VP64, shuttles to the nucleus, binds Plant:Pho and activatesexpression of the β-glucuronidase reporter gene. These observations showthat adaptive horizontal gene transfer can be used to produce syntheticeukaryotic signal transduction pathways.

The D53 in PhoB, that is fused with GFP, was mutagenized (Asp 53 toAla), the DNA sequence verified, and then transferred into Arabidopsisplants (FIG. 3D). PhoB^(D53A):GFP was assayed for function in plants byactivating the histidine kinase signaling pathway with cytokinin. Inmost cells PhoB^(D53A):GFP did not accumulate in the nucleus in responseto the cytokinin signal (FIG. 3D). There was some accumulation in cellsof the vascular tissues. However, these cells accumulate some PhoB:GFPwithout an exogenous signal (FIG. 3A) and it is possible that thebacteria response regulator in plants uses residues in addition to theD53 for shuttling. Like PhoB, mutagenesis of the bacterial conservedaspartate in OmpR also disrupts nuclear shuttling. FIG. 3D shows thatOmpR^(D55A):GFP did not show nuclear localization in response to theexogenous cytokinin signal. For PhoB, it was also shown that the Asp53residue is important in quantitative GUS assays with the TNT sensorprotein and hybrid HKs described below. FIGS. 3D and 3E show thatbackground (control) levels of TNT induction were close to 4 nM MU/mgprotein/hour whereas induced levels are consistently at 6 nM MU/mgprotein/hour or better (FIG. 3D). FIG. 3E shows that in plantscontaining PhoBD53A:VP64 signaling to activate expression of GUS, levelsare significantly less than 4 and only one plant (out of twenty) hadlevels approaching that of the control.

These observations show that adaptive horizontal gene transfer can beused to produce synthetic eukaryotic signal transduction pathways. PhoB,upon phosphorylation, has both DNA binding and transcriptionalactivation capabilities via its binding to “Pho Boxes” in the promoterregion of phosphate-responsive genes in the Pho regulon (VanBogelen etal. 1996). The crystal structure of bound PhoB (Blanco et al. 2002)showed that PhoB binds to a Pho Box as a homodimer as well as tomultiple Pho boxes in tandem. In the non-phosphorylated form, PhoB'sreceiver domain functions as a repressor, preventing the DNAbinding/transactivation domain from functioning.

Although PhoB has both DNA binding and transcriptional activationfunctions, the differences between prokaryotic and eukaryotictranscriptional activation are considerable. Without wishing to be boundby theory, it is believed that the PhoB transcriptional activationfunction would not work in a plant; therefore, the VP64 transcriptionalactivator was added to the C-terminal end of PhoB as a translationalfusion. The PhoB DNA binding and transcriptional activation activitiesoverlap in the bacterial protein and hence we did not believe they couldbe easily separated. VP64 is a transcriptional activator domainconsisting of four copies of the well-characterized VP16 activationdomain (Triezenberg et al. 1988 Genes Dev. 2:730-742; Triezenberg et al.(1988) ibid. 71-8-729), and was added to the C-terminal region of PhoBto create a synthetic protein PhoB:VP64. This synthetic protein wasengineered to be expressed in plants with a strong constitutive promoter(FMV, figwort mosaic virus promoter) and a common 3′ termination signal.A nopaline synthase (Nos) transcription termination sequence was addedat the 3′ end of the chimeric gene. The DNA binding consensus sequencefor PhoB is CTGTCATAYAYCTGTCACAYYN (SEQ ID NO:14).

Other Possibilities for the Response Regulator Protein:

Response regulator proteins are modular with a receiver domain that isphosphorylated and an effector domain which has DNA binding activity.These two domains are joined by a linker region, which varies amongdifferent response regulators. These linkers have been shown to beimportant for proper activity in bacteria (Walthers et al., 2003). Afusion of a receiver domain linker region (which includes but is notlimited to OmpR's receiver domain linker region) with an effector domain(which includes but is not limited to PhoB's effector domain, whichessentially has the DNA binding activity to the PlantPho promoter) ismade. It is expected that OmpR's receiver domain will be phosphorylatedmore efficiently (when compared to PhoB) by Trz. It is assumed thatOmpR's receiver domain will still be able to interact with PhoB'seffector domain and the protein will shuttle to the nucleus and bind thePlantPho promoter.

In another example, specific amino acids will be modified in a receiverdomain (which includes but is not limited to PhoB) to enable thereceiver domain to interact more effectively with Trz. These amino acidmodifications include, but are not limited to, T103P and S107N. Theamino acids that are modified include, but are not limited to, aminoacids that are responsible for the specificity of the interactionbetween the response regulators and their respective histidine kinases(which include, but are not limited to PhoR and EnvZ) (Li et al 2003).Once the amino acids have been modified, transgenic plants are generatedthat contain a target substance of interest receptor (which includes,but is not limited to ssTNT), a histidine kinase (which includes, but isriot limited to FLS-Trz), a response regulator with a modified receiverdomain (which includes, but is not limited to PhoB^(T103P,S107N):VP64)and a PlantPho promoter (which includes, but is not limited toPlantPho:Gus). It is expected that by modifying specific amino acids inPhoB (or any other receiver domain) to amino acids found in a cognatereceiver domain (such as OmpR), the interaction of the new receiverdomain with Trz will be improved.

Because of the high amount of evolutionary conservation of the responseregulators and histidine kinases in bacteria, fungi, cyanobacteria andplants, other response regulators and functional histidine kinases willalso work. The response regulators and the histidine kinases can be thecognate components from a specific system and/or functional componentsthat transmit a phospho-relay in plants.

PlantPho Promoter:

The PlantPho promoter was designed to allow for signal dependent PhoBtranscriptional activation in plants. The DNA binding consensus sequencefor PhoB is CTGTCATAYAYCTGTCACAYYN (SEQ ID NO:14). Eight tandem PhoBbinding sequences (TGTCA) were linked to a plant transcription startsite, the −46 region from the CaMV 35S promoter. The PhoB boxes and −46promoter, collectively constitute the “PlantPho promoter” (SEQ ID NO:1).

To prove this functions, the synthetic PlantPho promoter was placedupstream of and operably linked to various genes. These various genesinclude laboratory reporter genes such as the Green Fluorescent Protein(GFP) or β-glucuronidase (GUS), or the wide scale readout reporter genesand constructs of the degreening circuit. It could also include othergenes involved in the induction of a specific process such as floweringor production of a metabolic or pharmaceutical compound. These genes arecloned together with the FMV::PhoB-VP64-Nos construct into a planttransformation vector. Transgenic Arabidopsis plants with one simpleinsert were obtained. In the sentinel plant this promoter is operablylinked to at least one chlorophyll degradation enzyme coding sequence,and also desirably to an antisense or diRNA determining region forinhibiting expression of a chlorophyll biosynthetic enzyme.

In the sentinel plants of the present invention, the PhoB:VP64 movesinto the nucleus in response to the binding of the target substance tothe sensor portion (or ligand binding domain) on the exterior of thecell. In a model system, we looked for PhoB:VP64 to bind to the PlantPhopromoter and activate GUS or GFP expression. Plants were incubated forapproximately 16 h in the presence or in the absence of the ligand, of0.5 μM t-zeatin (a cytokinin).-Cytokinin-dependent induction of GUSexpression of up to 7.8-fold was observed. The data indicated thatPhoB:VP64 (synthetic response regulator designed from bacterialcomponents and with eukaryotic transcriptional activator) could directexternal signal-dependent expression from the synthetic PlantPhopromoter.

The cytokinin dependent induction showed that the signaling componentsworked and that horizontal gene transfer can be used to produceartificial signal transduction pathways in higher eukaryotic organisms.To link this synthetic signal transduction pathway with input from thecomputationally designed receptors, the extracellular targeted sensorprotein was added (FIG. 1) and separately, the various hybrid histidinekinases shown in FIG. 2. To prove this system functions, perception ofthe target substance (TNT) was linked to quantitative expression of theGUS reporter gene.

FIG. 4A-C show that three separate signaling systems all function intransgenic plants to report the presence of the TNT ligand. In FIG. 4A,plants are exposed to TNT, TNT binds the sensor protein, the sensorprotein develops affinity for the exterior of the histidine kinase(Trg), the histidine kinase is activated, and the operably linkedinterior AHK4 transmits a high energy phosphate group to AHPs, that thenshuttles to the nucleus and activates transcription of a Type B ARR gene(such as ARR5).

In FIG. 4B, plants are exposed to TNT, TNT binds the sensor protein, thesensor protein develops affinity for the exterior of the histidinekinase (Trg), the histidine kinase is activated, and the operably linkedinterior (PhoR) transmits a high energy phosphate group to PhoB:VP64that then shuttles to the nucleus and activates transcription of thePlantPho promoter linked to GUS. FIG. 4B also shows detailed statisticaldata (ANOVA) indicating that the data are significant. All assumptionsfor this analysis were tested and met. In addition, FIG. 4B shows thatthe system is responding to extremely low levels, 10 picomoles, of TNT.

In FIG. 4C, plants are exposed to TNT, TNT binds the sensor protein, thesensor protein develops affinity for the exterior of the histidinekinase (Trg), the histidine kinase is activated, and the operably linkedinterior (EnvZ) transmits a high energy phosphate group to PhoB:VP64that then shuttles to the nucleus and activates transcription on thePlantPho promoter linked to GUS.

In FIG. 4D, the eukaryotic activation domain (VP64) is replaced with asmaller version, VP16. Cytokinin induction or cross kingdom signaling(with cytokinin induction) shows that other eukaryotic activationdomains can also work in addition to VP64. GUS induction is somewhatbetter with VP16 than VP64.

Shuttling Protein/Response Regulator:

Hybrid histidine kinases containing both bacterial and plant segmentswere unexpectedly shown to be functional in both bacteria and plants.This discovery is important because all hybrid histidine kinases to dateare ones made from portions of similar bacterial histidine kinases. Inaddition, it provides a molecular foundry to rapidly move genes betweenbacteria and plants, opening the door for directed evolution approaches.

In a specific example, a hybrid was created between the bacterialperiplasmic histidine kinase (TRZ) and the plant histidine kinase (AHK4)normally found within the plant cell membrane. Hence, when the hybridhistidine kinase is activated by binding of a ligand bound receptor, thesignal is transmitted via a phosphor-relay to an AHP via phospho-relaythat moves into the nucleus and activates transcription (ARR5 or ARR7promoter).

Quantitative (GUS) and qualitative (GFP) data were obtained fortransgenic Arabidopsis plants homozygous for the ARR5 promoter fused toGUS and GFP, respectively, retransformed with the computationallydesigned receptor for TNT (SS:TNT) and the hybrid histidine kinase(plant leader (FLS):TRZ:AHK4). In response to TNT, the ARR5 promoter wasactivated causing an approximate 2-fold increase in GUS expression.

Components of the pathway were separately deleted. When the histidinekinase was deleted, no induction was seen and when the TNT receptor wasdeleted, no induction was observed. Our experimental data show that wecan build a synthetic signal transduction that functions in plants frombacterial and designed components.

The pathway shown to function is FLS:TRZ:AHK4→AHP→type B ARR→type A ARRpromoter.

The coding sequence of the TRG receptor (exterior) with EnvZ (interior)is assembled via molecular biological techniques (Baumgartner J M, etal., 1994). The EnvZ on the membrane interior sends a phospho-relay toits cognate response regulator OmpR and also to PhoB. The bacterialresponse regulator PhoB's cognate histidine kinase is PhoR. Twofunctional fusions of TRG (exterior) to PhoR interior have been made andshown to function in bacterial cells (SEQ ID NO:2, SEQ ID NO:3 and SEQID NO:12). They were then incorporated in a complete signaling pathway(with the designed receptors) in plants. The complete signaling pathwayin this case is: SS-TNT→FLS:TRG:PhoR→PhoB:VP64→PlantPho promoter:GFP orPlantPho promoter:GUS.

The sensing by the computationally designed receptor for TNT occurs whenSS-TNT binds TNT, then develops high affinity for and binds Trg. BindingTrg activates the histidine kinase to start a phospho-relay system toPhoB:VP64. Upon phosphorylation, PhoB:VP64 moves into the nucleus, whereit binds to the synthetic PlantPho promoter and activates transcriptionof the GFP or GUS gene in the demonstration project, and the degreeningcircuit in the sentinel plants of the present invention. It could alsoactivate expression of any type of plant response gene typical for plantbiotechnology such as those involved in flowering or those involved ininitiating a metabolic or pharmaceutical pathway. In the degreeningcircuit, the synthetic PlantPho promoter is operably linked to thecoding sequence of a chlorophyll degradation enzyme such aschlorophyllase, and desirably a synthetic PlantPho promoter is alsooperably linked to sequences encoding antisense or small interfering RNAspecific to at least one chlorophyll biosynthetic enzyme.

T0 represents the first generation of transgenic plants whereas T1represents the second generation (T0 seed self-pollinated). Seed fromtwo of the T0 parental lines has been obtained. These lines segregatefor the two T-DNA inserts that contain the signaling components. OneT-DNA contains the genes for the receptor and histidine kinase (SS-TNT,FLS:Trg:PhoR) while the other T-DNA contains the genes for the responseregulator (PhoB:VP64) and the PlantPho promoter fused to GUS(PlantPho:GUS). In bacterial sensing and signaling systems, thesensitivity and response systems are extremely sensitive to the other'slevels. In the transgenic plants, the two genetic sets segregate in aclassic 9:3:3:1 pattern.

It is possible to substantially improve the sensing and signalingsystems by modulating the expression levels of receptor/HK and signalingcomponents (PhoB:VP64, PlantPho promoter) via expression controls(promoter, translational enhancer etc.) and via copy number. Microarraydata from the degreening circuit indicate the response required aneight-fold increase in the expression levels of the degreening circuitgenes' expression levels. The data from FIG. 4 shows that there is abouta two-fold increase in expression with sensing TNT in model plants. Thedegreening system is linked to the Pho (synthetic) sensing system (thesentinel plants) and provides similar sensitivity to the targetsubstance for which the specific binding site is engineered into thesensing portion of the input circuit. Specifically, the 10XN1 Plantpromoter driving the degreening circuit genes is replaced by thesynthetic Pho promoter. Because the system is modular, the TNT receptorcan be replaced with any designed specific receptor site for a targetsubstance of interest. It is important to note that the sensing systemhas been shown to work with two independent signaling systems. Thedesigned receptors provide an extremely high level of sensitivity(nanomolar) and extremely high specificity, as demonstrated in thebacterial system (Looger et al., 2003)

Protochlorophyllide oxidoreductase (POR) is the key enzyme inchlorophyll biosynthesis (Malkin and Niyogi, 2000). Arabidopsis containsthree genes for POR (Oosawa et al., 2000; Pattanayak and Tripathy,2002). To block expression of all three AtPOR (Arabidopsis thaliana POR)genes, a diRNA construct to a region conserved in each of the genes wasproduced. A conserved region of approximately 500 bp was identifiedlocated from nucleotides 700-1200 within the AtPOR 1708 bp cDNA that isconserved in all three genes. Standard PCR methods were used to amplifythis conserved region from a full-length cDNA (AT1G03630). To facilitatecloning, forward and reverse primers with restriction sites were usedfor AscI and BamH1 in the forward primer and SwaI and XbaI on thereverse primer. The target DNA in POR genes lacks the sites for theserestriction endonucleases. The conserved region of the POR gene wasamplified and its sequence was verified by sequencing. The PCR productwas then digested with AscI and SwaI and ligated to AscI-SwaI-cleaveddsRNA vector. Subsequently, the PCR product was digested with BamH1 andXbaI and ligated into flanking sequences of the vector to produce aconstruct that will express an inverted repeat of the conserved regionof the POR genes. By doing so, expression from all three POR genes canbe silenced by the dsRNA produced.

Chlorophyllase:

The complete chlorophyll degradation pathway is known to the art. Toproduce the degreening circuit, two key genes in this pathway wereinduced. One of the key enzymes involved in chlorophyll breakdown ischlorophyllase (Matile et al., 1999; Tsuchiya et al., 1999; Benedettiand Arruda, 2002). Constitutive expression of the chlorophyllase gene(AtCOR1) leads to a massive accumulation of the breakdown productchlorophyllide (Benedetti and Arruda, 2002). Because the chlorophyllideproduct still has a green color (light green for chlorophyllide versusdark green for chlorophyll), a second gene was included to furtherdegrade the breakdown product. The next gene in the chlorophyllbreakdown pathway is magnesium dechelatase that removes the Mg from thefour-ring structure. Action of this enzyme leads to formation ofpheophorbide a, a molecule that still retains some of the green color(Dangl et al., 2000). Because of this, one of the two enzymes involvedin opening the ring structure was used to eliminate the remaining greencolor. In chlorophyll breakdown, ring opening occurs by the joint actionof pheophorbide a oxygenase (PAO) and red chlorophyll catabolitesreductase (RCC reductase) (Matile et al., 1999). Intracellularly, PAOand RCC reductase are thought to be juxtaposed with PAO located on theinner membrane of the chloroplast envelope and RCC reductase locatedjust inside in the stroma (Matile et al., 1999). While the Arabidopsisgenome contains several candidates for PAO, biochemical proof that thesegenes encode PAO is lacking. In contrast, the Arabidopsis RCC reductasegene has been isolated and characterized (Wuthrich et al., 2000; Mach etal., 2001). Therefore, RCC reductase was used in the present work. Boththe chlorophyllase gene (AtCOR1) and RCC reductase (NCBI Accession No.Z99707) can be placed under control of promoters that induce expressionin response to input from either cytoplasmic or extracellular analytes.

Rapid degreening requires the inhibition of chlorophyll synthesisconcurrent with the induction of degradation. Accordingly, we firstassembled each function separately under control of thesignal-responsive transcriptionally inducible system. Two means toinhibit net chlorophyll synthesis (“stop synthesis” circuits) were used:directly, through its biosynthetic pathway; or indirectly, through aprecursor/trafficking pathway. The rate-limiting enzyme in chlorophyllbiosynthesis is NADPH:protochlorophyllide oxidoreductase. In Arabidopsisthis enzyme is encoded by three POR genes (PORA, PORB, PORC) (Frick etal., 2003; Masuda et al., 2003; Oosawa et al., 2000). POR is alight-dependent enzyme that catalyzes the conversion ofprotochlorophyllide a (Proto) to chlorophyllide a. GUN4, GenomesUncoupled 4, is a single copy gene that functions in regulatingchlorophyll biosynthesis, precursors trafficking, and may have a role inphotoprotection (Larkin et al., 2003; Verdecia et al., 2005). GUN4 hasbeen shown to bind and activate Mg-chelatase, an enzyme complex thatproduces Mg-protoporphyrin IX (MgProto). A diRNA construct was preparedto a conserved POR gene region, its expression placed under control of asignal-dependent inducible promoter, and the construct introduced intoArabidopsis plants. A diRNA construct to GUN4 was also prepared, andlikewise placed under control of the inducible promoter. Asignal-dependent induction of the diRNA to POR or GUN4 alone did notcause plants to lose their green color.

Genes encoding key enzymes in chlorophyll breakdown have also beenidentified (Eckhardt et al., 2004). Chlorophyll breakdown involves aseries of enzymatic steps, with key processes being hydrophobic tailremoval by chlorophyllase (CHLASE) (Benedetti and Arruda, 2002; Tsuchiyaet al., 1999), porphyrin ring cleavage by PAO (pheide a oxygenase) andsubsequent action of RCCR (red chlorophyll catabolite reductase)(Pruzinska et al., 2003; Pruzinska et al., 2005; Wuthrich et al., 2000).Two distinct gene circuits designed to initiate chlorophyll degradationwere assembled (“initiate breakdown” circuits), containing CHLASE andeither PAO or RCCR under control of the signal-dependent promoter. Evenafter 48 hours of induction of the chlorophyll “initiate breakdown” theplants also remained green, despite the over-expression of the two majorgenes involved in chlorophyll metabolism. These results are consistentwith our hypothesis that plants are able to partially compensate forchanges in chlorophyll levels by regulating overall metabolism.

“Stop-synthesis” constructs with the “initiate breakdown” genes werecombined in one T-DNA to test if a rapid, regulated chlorophyll losssystem could be developed. The constructs/genes were brought together invarious combinations to produce 5 different “degreening gene circuits”(Table 1, FIG. 5A). Each “degreening circuit” consists of an induciblediRNA to either POR or GUN4 genes, CHLASE inducible expression, combinedwith PAO and/or RCCR inducible expression. Degreening circuits were alsoobtained by crossing plants containing the separate gene circuits andproduced comparable results.

TABLE 1 Genes used in constructing each of the complete degreeningcircuits Degreening Stop Initiate Circuit # Synthesis Breakdown 1 PORCHLASE, RCCR 2 GUN4 CHLASE, RCCR 3 POR CHLASE, PAO 4 GUN4 CHLASE, PAO 5GUN4 CHLASE, RCCR, PAO

FIG. 5A shows that induction of the degreening circuits caused a loss ofchlorophyll from all regions of the plant, with less effect early on inthe shoot apex. Chlorophyll loss is extensive with plants becoming whitewithin 24-48 hours after induction. In senescing Arabidopsis, loss ofchlorophyll typically reveals yellow carotenoid pigments; the whitephenotype observed upon induction of the synthetic degreening circuitssuggests that carotenoid pigments are also lost.

A notable feature of chlorophyll loss by the degreening circuits is thatthe rational design of the synthetic circuit (simultaneous regulation ofbiosynthesis and breakdown) leads to a similar white phenotyperegardless of the specific gene combination. Only a slight differencewas seen between stopping synthesis with diRNA to POR and diRNA to GUN4(compare circuit #1 to #2, or circuit #3 to #4). Inclusion of the RCCRgene with CHLASE to initiate breakdown produced slightly betterchlorophyll reduction than breakdown initiation that involved PAO andCHLASE (compare circuit #1 and #2 to circuit #3 and #4). Degreeningcircuit #5 differs from the others as it contains the PAO and RCCR (aswell as CHLASE) genes to initiate breakdown. Plants containing circuit#5 lose chlorophyll within 24 hours; however, the plants are light greenprior to induction.

Changes in the expression of degreening circuit genes were verified withsemi-quantitative RT-PCR (FIG. 5B). As predicted, induction of diRNAconstructs lead to decline in the respective mRNAs: POR mRNA declined inplants with circuits #1 and #3; GUN4 mRNAs declined in circuit #2 and toa lesser extent in circuits #4 and #5. mRNAs for genes that are inducedwere found to dramatically increase: CHLASE and RCCR increased in plantscontaining circuit #1 or #2. For plants containing circuit #3 or #4,CHLASE levels increased and PAO induction showed a strong increase incircuit #3, with a more modest induction in plants containing circuit#4. Plants containing circuit #5, which were light green prior toinduction, appear to have lost regulation of the CHLASE and RCCR genes.Levels of cyclophilin mRNA were used as a control and in some linesthese levels declined, likely reflecting the decline in total RNA at 24and 48 hours of induction. Consequently, the RT-PCR results likelyunder-estimate reductions/increases in levels of degreening circuit genemRNAs. This is confirmed by analysis of CHLASE in circuit #3; RT-PCRresults indicate a 3-fold induction, whereas microarray analysis showedit was induced >8 fold.

To determine if decreased levels of POR and GUN4 transcripts producedcorresponding decrease in the proteins, Western blots using antibodiesto POR and GUN4 were performed. Induction of the degreening circuits ledto dramatic decrease in POR and GUN4 proteins, with levels decreasing by70-90% within 24 hours.

Reset capacity is an essential feature both in a plant functioning as asentinel for terrorist threats, to allow multiple or repeated threats tobe detected, and as a plant environmental monitor for long-termpollutant monitoring. Attempts have been made to develop resetcapacities in plant reporter genes by decreasing mRNA stability. The useof the degreening circuit as a reporter system provides an endogenousmeans to reset the system. Plants that had lost their chlorophyll frominduction of the degreening circuit re-developed their green color orregreened after the inducer was removed (FIG. 5A, Regreen). Theregreening process was enhanced with cytokinin treatment, providing asimple and readily available means to reset the reporter system.Furthermore, regreened plants can be induced to degreen again.Regreening is most apparent in rapidly expanding leaves. However,partially degreened leaves and tissues are also capable of regreening.Older leaves that completely degreened early in the process, and havelost turgor, did not regreen. In regreening plants, the levels of PORand GUN4 transcripts increased, while levels of CHLASE, PAO, and RCCRmRNAs decreased to levels approaching normal (FIG. 5B).

Because light plays an essential role in chlorophyll metabolism, weasked if light is required for degreening. Plants grown under differentlight intensities (50 to 350 μE·m⁻²·s⁻¹) and different temperatures (14°C. to 30° C.) were not affected in their ability to degreen. However,when plants were grown under standard light conditions and induced todegreen in complete darkness, the effects of the degreening circuit weremuted (FIG. 5E, part a). After 24, 48 or 72 hours of induction incomplete darkness the plants eventually pale, but are still a lightgreen, indicating that light is required for rapid chlorophyll loss. Ifplants induced in complete darkness were subsequently transferred to thelight, degreening proceeded at an enhanced rate.

In Arabidopsis, detached leaves and whole plants show a distinctresponse to dark-induced senescence (Weaver and Amasino, 2001). Darknessis known to induce senescence in detached leaves but not in wholeplants. To determine if the degreening circuit functioned similar tosenescence we induced degreening in detached leaves. As expected, understandard light conditions degreening induction caused detached leaves tofully degreen within 48 hours (FIG. 5E, part b). However, darkness byitself failed to induce full degreening in detached leaves even after 72hours (FIG. 5E, part c). We conclude that the degreening circuit isinducing a synthetic pathway that is distinct from chlorophyll lossfound in senescence.

Signal-induced chlorophyll loss provides an easily recognizablephenotype (white plants) that is distinct from stressed plants, and areset capacity. The two remaining characteristics needed for a readoutsystem in a plant sentinel are rapid response and remote imagingcapacity.

Light energy absorbed by chlorophyll follows several competing paths:photosynthetic electron transport drive, heat dissipation through thexanthophyll cycle, re-emission as fluorescence, or formation of tripletchlorophyll (Maxwell and Johnson, 2000). Because the pathways arecompeting, and the sum of rate constants is unvarying, information aboutthe plants physiological state can be inferred by remote analysis ofchlorophyll fluorescence (West et al., 2003; Zarco-Tejada et al., 2002).In addition, because plants must quickly respond to changing lightconditions, chlorophyll responses are rapid.

To determine if chlorophyll fluorescence measurements would providerapid diagnostic and remote imaging capacities to the degreening circuitsystem, we used commercially available chlorophyll monitor and software(Fluorcam, Photon Systems Inc.) to measure how chlorophyll fluorescencebehaved before and after induction of the degreening circuit.

Control plants, both induced and uninduced, showed only slight changesin all parameters measured (FIG. 5C). F_(v)/F_(m) measures the maximumquantum efficiency of PSII in dark-adapted plants and is often used as ameasurement of plant stress (Maxwell and Johnson, 2000). Uninducedplants containing the degreening circuit had an initial F_(v)/F_(m)value of 0.8 (FIG. 5C bottom). An F_(v)/F_(m) value of 0.8 is close tothe maximum possible PSII efficiency observed in normal, non-stressedplants (Adams et al., 1990). These results indicate that there arelittle physiological effects of the presence of the (silent) degreeningcircuit in uninduced plants. Upon induction of the degreening circuit,F_(v)/F_(m) declines. Initial declines in F_(v)/F_(m) were seen within 2hours, and more substantial reductions observed at 6 hours.

F_(m) is a measure of maximum chlorophyll fluorescence and an indirectmeasurement of total chlorophyll in dark-adapted plants. Afterdegreening circuit induction, F_(m) showed some initial variability,with stable reductions seen after 12 hours (FIG. 5C top). These initialvariations in F_(m) are similar to those found when total chlorophylllevels were measured spectrophotometrically (FIG. 5C top). Withoutwishing to be bound by any particular theory, this is believed toreflect plant mechanisms to attempt to compensate for enhancedchlorophyll degradation.

One of the most robust parameters derived from chlorophyll fluorescencemeasurements is φ_(PSII), representing the portion of light absorbed bychlorophyll in photosystem II (PSII) used in photochemistry. φ_(PSII)can also be measured in light grown plants under natural conditions(Maxwell and Johnson, 2000). FIG. 5C (middle) shows that plants inducedto degreen had a large (nearly 60%) reduction in φ_(PSII) within 2hours, the first time point measured. These results show the measurementof φ_(PSII) provides a straightforward means to rapidly and remotelydetect induction of the degreening circuit reporting system.

Global changes in gene expression during degreening are distinct fromthose of senescence. The degreening circuit causes chlorophyll lossrelatively quickly, with response of leaves to dark treatment andultrastructural aspects distinct from normal chlorophyll loss observedduring senescence. To further test the idea that the degreening circuitinitiates a synthetic process distinct from the chlorophyll loss insenescence, we employed microarray analysis. If the degreening circuitinitiates a synthetic process, the genes involved in senescence shouldbe relatively unaffected while genes encoding photosystem componentsmight be affected. FIG. 5F shows plots comparing major classes of genesdown-regulated by the degreening circuit to those down-regulated bysenescence. As expected, the majority of annotated PSII- and PSI-relatedgenes (e.g., light-harvesting chlorophyll a/b binding andoxygen-evolving complex proteins) are down-regulated within 24 hours ofinduction, the first time point measured. This includes PSII subunits(PSO1, PSO2, PSP1, PSP2, PSQ1, PSQ2) and PSI subunit precursors(Kieselbach and Schroder, 2003). Other notable down-regulated genesinclude DegP2, encoding a protease that is responsible for initialrepair of damaged PSII proteins (Haussuhl et al., 2001), and FtsH6, achloroplast LHCII protease (Zelisko et al., 2005). After 48 hours, thesegenes are also down-regulated. Also notable among the groupdown-regulated by the degreening circuit is PsbS (NPQ4), which allowsexcessive energy to be dissipated by photosynthetic antennas through thexanthophyll cycle (Holt et at., 2005; Li et al., 2004). While mostphotosystem genes are down-regulated, a few known photosystem genes werefound to be up-regulated, including PsbP and genes encoding lumenproteins of unknown function (e.g., At1g03600, At3g09490).

In addition, microarray analysis shows that genes involved in ROS areup-regulated by the degreening circuit. Genes involved in anti-oxidativeprocesses (Mittler et al., 2004), and genes involved in detoxifyingproducts of lipid peroxidation (Loeffler et at., 2005) are induced bydegreening (redox regulation and oxidative stress), includingglutathione transferase, microsomal glutathione S-transferase, type 2peroxiredoxin, NADP-dependent oxidoreductase, glutaredoxin, thioredoxin,peroxiredoxin, alternate oxidase, ferritin, blue copper protein,glutathione peroxidase, and several other peroxidases. The majority ofgenes known to be up-regulated during senescence were not induced by thedegreening circuit (FIG. 5F). Genes that are strongly induced duringsenescence, but not during degreening, include enzymes for degradationof macromolecules (e.g. proteases, nucleases, lipases), transcriptionfactors, kinases/phosphatases, defense related genes andflavonoid/anthocyanin biosynthetic genes (Buchanan-Wollaston et al.,2005). Within certain categories, small subsets of genes were commonlyinduced by both senescence and degreening, including chaperones, redox,autophagy, and alkaloid-like biosynthetic genes. Genes down-regulated insenescence were likewise largely not induced along with the degreeningcircuit. The prominent exception, where down-regulated senescence geneswere down-regulated in degreened plants, is most of the nuclear encodedcomponents of the photosynthetic machinery, including PSII, PSI, andCalvin Cycle genes.

Computational Designed Receptors Used to Activate Degreening Circuit:

In order to demonstrate that the signal transduction pathway activatedvia computational designed receptors can activate the degreening circuitthe 10XN1P promoters from the model degreening circuit were replacedwith PlantPho promoters. Arabidopsis and tobacco plants were thenco-transformed with the PlantPho promoter-degreening circuit and theSS-TNT, FLS:TRG:PhoR, PhoB:VP64 construct. FIG. 6 shows that bothArabidopsis and tobacco leaves incubated in the presence of TNT showed adegreening phenotype. FIG. 6A shows that the degreening circuit isinduced with 100 μM (23 parts per trillion) of TNT in transgenicArabidopsis plants resulting in white leaves. FIG. 6C shows thisinduction could be detected within 5 hours using remote techniques.FIGS. 6A and 6C show that a functional plant sentinel can be built.FIGS. 6B and 6D show that the technology also works in tobacco. FIG. 6Bshows that notable degreening in these plants was induced with 10 μM TNTand the remote readout (chlorophyll fluorescence) was notably differentat 24 hours.

The above studies demonstrate that the degreening circuit of theinvention can exhibit the degreening phenotype specifically andselectively in response to a signal. The loss of green color is quick,sensitive, and easy to detect directly or by remote sensing.Furthermore, the components of the degreening circuit are modular inthat each component can be replaced with a specific component (e.g.binding protein/receptor or promoter) to provide a specific andselective response for a given input signal (target substance). FIG. 7shows an example where the input (using for example TNT) activatestranscription of a specific gene such as the FT (Flowering Locus T) genecritical for the conversion to flowering. Because of the modularity inthe system, the input control could readily be changed so that, forexample, the FT gene is induced by glyphosate, ribose or any of thecomputationally designed receptors.

FIG. 8 shows enhancements to the degreening circuit that were evidentfrom the microarray analysis. Both the DegP2 and FtsH gene encodesproteases involved in photosystem repair. Both genes have alteredexpression during the degreening process. By directing their expressionwith the PlantPho promoter the degreening readout system is faster andmore independent of light.

FIG. 9 shows details for making a “trigger” circuit that will allowresponse to a single exposure to a specific ligand. The featuresdescribe a series of feedback genes (repressors) that are tuned withsynthetic biology for proper function. Critical to the setting processis using the fact that genes are not transcribed in the male pollenwhereas they are transcribed in the female egg sac.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

Experimental Details

Plant materials, growth conditions and transgenic plant production.Arabidopsis thaliana, ecotype Columbia (Col-0) plants were used foranalysis and transgenic plant production. Standard growing conditionswere 25° C., approximately 100 μE m⁻²·s⁻¹ light in either a PercivalAR75L growth chamber (25°±1° C.) or light shelf (25°±2° C.), with aday/night cycle (16 h light, 8 hour dark), in Metro Mix 200 growth media(Scotts, Marysville, Ohio), supplemented with MIRACLE-GRO fertilizer.Transgenic plants were produced using the floral dip method (Clough andBent, 1998). Plasmids were assembled as described below, transferredinto Agrobacterium tumefaciens strain GV3101 by electroporation, and theselected Agrobacterium used for Arabidopsis transformation. Transgeniclines were selected on Murashige and Skoog (MS) media (Murashige andSkoog, 1962) containing 50 mg/L kanamycin or 5 mg/L glufosinate(Crescent Chemical Co., Islandia, N.Y.), depending on the vector, and100 mg/L cefotaxime. T₀ lines were selected and allowed toself-pollinate, and only lines segregating for one T-DNA insert (3:1)and not showing any obvious phenotypic lesion were analyzed further.When possible, homozygous transgenic lines were obtained and used foranalysis (degreening circuits #1, #2, and #3). With two degreeningcircuits (#4 and #5), homozygous lines could not be obtained or thehomozygous lines set seed too poorly for detailed analysis. For plantscontaining these circuits, heterozygous lines were used.

Chemicals and Enzymes. MS salts, vitamins, and 4-hydroxytamoxifen(4-OHT) were all purchased from Sigma-Aldrich (St. Louis, Mo.). 5 mM4-OHT stock solutions were prepared in ethanol and stored at −20° C.Enzymes used for cloning and DNA amplification were obtained fromcommercial suppliers and used according to the manufacturer'sinstructions.

Plasmid Construction. To test and develop the model degreening genecircuits, a chemically inducible transcription system was assembled forplants using the synthetic zinc finger proteins and DNA binding elementsas described by Barbas and colleagues (Beerli et al., 2000; Segal etal., 2003). This steroid-regulated transcription system in plants isvery similar to estrogen inducible systems previously described (Zuo etal., 2000), and use of these components in plants has been previouslydescribed (Stege et al., 2002).

The regulatory elements for the inducible system were first assembled. Asteroid binding chimeric transcription factor (NEV) was placed undercontrol of a strong constitutive promoter (FMV, figwort mosaic virus)(Bhattacharyya et al., 2002; Sanger et al., 1990), translation enhancedwith the omega enhancer at the 5′ end (Gallie et al., 1987), andexpression terminated with the 3′ end of the Nos (nopaline synthase)gene. NEV is a fusion protein, containing the synthetic NI zinc fingerDNA-binding domain (N), an estrogen receptor domain (E), and four copiesof the herpes simplex virus (VP16) transcriptional activation domain (V)(Beerli et al., 2000). In animal cells, the NEV protein translocates tothe nucleus and binds the synthetic N1 DNA element in the presence ofthe inducer. To make this system functional in plant cells, wesynthesized the 10 copies of the N1 DNA element upstream of the minimal−46 CaMV35S promoter (Odell et al., 1985) (Egea Biosciences, Inc,. SanDiego, Calif.), creating a 223 bp promoter fragment referred to as10XN1P. Both the chimeric transcription factor (FMV::Ω-NEV-Nos) and thesynthetic N1 promoter 10XN1P were cloned into the plant transformationvectors pCAMBIA2300 or pMLBART.

To produce a synthetic degreening circuit, genes regulating chlorophyllbiosynthesis and breakdown were placed under control of the inducible10XN1P promoter and introduced into plant transformation vectors.Because multiple genes (up to 4) were introduced, a transcription blockwas placed between each gene or the genes were placed in a manner so asto prevent interference with expression of those genes (Padidam and Cao,2001). The complete degreening circuit combines two types of geneticcircuits: a gene circuit to inhibit biosynthesis of new chlorophyllbiosynthetic enzymes, and thus chlorophyll synthesis, and a gene circuitto stimulate chlorophyll breakdown. All chlorophyll regulatory genes inthe different constructs were placed under control of the 10XN1Ppromoter, allowing a coordinated expression upon addition of the 4-OHTinducer.

To inhibit biosynthesis of chlorophyll, diRNA constructs were assembledspecific to the GUN4 gene (Larkin et al., 2003) or a conserved regionfound in the POR genes (Masuda et al., 2003; Oosawa et al., 2000). Togenerate the diRNA constructs, POR sense-intron-POR antisense, or GUN4sense-intron-GUN4 antisense were cloned into pBluescript KS(+),including approximately 500 bp GFP coding sequence used as intronsequences at NotI/EcoRV. POR sense/GUN4 sense was cloned in viaSacI/NotI and POR antisense/GUN4 antisense cloned in via EcoRV/XhoI(XmaI/XhoI for the GUN4 diRNA construct), then subcloned downstream of10XN1P in p2300-FMV::Ω-NEV-nos-10XN1P at the AvrII/MluI sites. Anoctopine synthase (ocs) terminator was added at the MluI site, resultingin p2300-FMV::Ω-NEV-nos-10XN1P::POR diRNA-ocs, orp2300-FMV::Ω-NEV-Nos-10XN1P::GUN4 diRNA-ocs.

The gene circuit to initiate the breakdown of chlorophyll contains theChlorophyllase1 (CHLASE) (Tsuchiya et al., 1999) gene to removechlorophyll's hydrophobic tail, and a gene or gene(s) to open theporphyrin ring, Red Chlorophyll Catabolite Reductase (RCCR) (Wuthrich etal., 2000) and/or Pheophorbide a Oxygenase (PAO) (Pruzinska et al.,2003). Chlorophyll degradation genes were added as a combination ofeither CHLASE and RCCR, or CHLASE and PAO; or, in its final form asCHLASE, PAO and RCCR. These gene combinations, all under control of the10XN1P promoter, were first assembled in pBluescript KS(+). CHLASE wasplaced downstream of 10XN1P via BstXI/NotI, and the Nos terminator wasadded at the SpeI/SmaI sites; RCCR was fused with 10XN1P via PstI/EcoRI,and the Nos terminator (EcoRI/ApaI). Transcription blocks (TB) wereincluded between the Nos terminator and 10XN1P via SmaI/PstI. The RCCRgene was replaced with PAO using the same restriction sites describedabove, to generate the CHLASE-PAO combination. Assembled10XN1P::CHLASE-Nos-TB-10XN1P::RCCR-Nos or10XN1P::CHLASE-Nos-TB-10XN1P::PAO-Nos was added to the 3′ end of eitherPOR or GUN4 diRNA in p2300-FMV::Ω-NEV-Nos-10XN1P::POR diRNA-ocs (orp2300-FMV::Ω-NEV-Nos-10XN1P::GUN4 diRNA-ocs), as an ApaI fragment, tocomplete degreening circuits. The “initiate breakdown” constructs(CHLASE+RCCR, and CHLASE+PAO) were assembled by first PCR-amplifyingFMV::Ω-NEV-Nos-TB from p2300-FMV::Ω-NEV-Nos-TB using primers FMVFwd,5′-ATTTAGCAGCATTCCAGATTGGGTTC-3′ (SEQ ID NO:15), and TBRev,5′-AGAGAAATGTTCTGGCACCTGCACTTG-3′ (SEQ ID NO:16). The PCR product wascloned as a blunt fragment into SpeI-digested and Klenow-treatedpART27-based vector pMLBART (Gleave, 1992), resulting inpMLBART-FMV::Ω-NEV-Nos-TB. The CHLASE and RCCR genes, as well as theCHLASE and PAO genes, all under control of the 10XN1P promoter wereexcised from vectors containing degreening circuits #1 and #3,respectively, as ApaI fragments. After flushing the ends, thesefragments were ligated into NotI-digested, Klenow-treatedpMLBART-FMV::Ω-NEV-Nos-TB. All gene fusions and all chlorophyllregulatory genes were verified by sequencing (Macrogen Inc., Seoul,Korea) before final assembly into pCAMBIA2300 and pMLBART vectors.

Generation of transgenic plants. ssTNT, FLS:Trz:AHK4 construct: A fusionbetween the Pex secretory sequence and the periplasmic ribose bindingprotein engineered to bind TNT (the procedure for this fusion will workon any engineered RBP) was formed using a variation of standard PCRtechniques. Primers were designed that created a sequence overlapbetween the secretory sequence and RBP. Two initial PCR reactions wereperformed using standard PCR conditions in 50 μl reactions. One reactionon the Pex secretory sequence using primers5′-CTTCGGATCCATGGAGAGGCCCTTTG-3′ (SEQ ID NO:17) and5′-CGCGATGGTGTCTTTGGCCACGACGGTATA-3′ (SEQ ID NO:18). The second reactionon the engineered RBP gene used primers5′-TATACCGTCGTGGCCAAAGACACCATCGCG-3′ (SEQ ID NO:19) and5′-AGGAGAGCTCTACTGCTTMCMCCAG-3′ (SEQ ID NO:20) An MJ thermocycler and ahigh fidelity polymerase (expand high fidelity Taq polymerase, Roche)were used. The products from the initial PCR contain overlappingsequence homology at the junction point between the secretory sequence(SS) and the RBP sequence. The PCR products were diluted to about 10ng/μl and placed in a standard PCR reaction mix which lacked anyprimers. The PCR reaction mix was subjected to a PCR cycle of 95° C. for2 min, 52° C. for 1 min and 72° C. for 3 min overall for 3 cycles. Thisallowed the overlapping sequences between the Secretory Sequence (SS)and RBP to anneal together. The annealed ends of the PCR products canthen act as primers to facilitate a reaction where the Taq polymerasereplicates or “fills-in” the rest of the gene fusion creating a doublestranded SS-RBP (or ssTNT) gene fusion. After 3 cycles the upper primerused on the Secretory sequence 5′-CTTCGGATCCATGGAGAGGCCCTTTG-3′ (SEQ IDNO:21) and the lower primer used on the RBP sequence5′-AGGAGAGCTCTACTGCTTAACAACCAG-3′ (SEQ ID NO:22) were added to the PCRtubes. This allowed for amplification of the full length SSTNT productafter 27 cycles using standard PCR conditions. Intermediate cloning ofthe PCR products was done using PCRTerminator end repair kit andCloneSmart kit vector pSMART from Lucigen and coding sequence integritywas confirmed by sequencing. The SS upper primer contains a BamH1 siteand the lower RBP primer contains a SacI site allowing cloning of thessTNT gene fusion into the BamH1 and SacI sites between the 35S promoterand Nopaline Synthetase Terminator of the plant transformation vectorpCB302-3. A similar procedure was performed to form a fusion between theFLS2 signal sequence and the Trz:AHK4 gene using the same techniquesdescribe above, and using primers 5′-GTTGCGGATCCATGMGTTACTCTCAAAG-3′(SEQ ID NO:23) and 5′-GATACGGTTMTCATTTTCGCTAGTGCMT-3′ (SEQ ID NO:24) forthe plasma membrane targeting sequence of FLS2 and primers5′-ATTGCACTAGCGAAAATGATTMCCGTATC-3′ (SEQ ID NO:25) and5′-GCGATCGCTTACGACGMGGTGAGATAG-3′ (SEQ ID NO:26) for Trz:AHK4.FIs:Trz:AHK4 was cloned into the BamHI and SgfI sites of a pBluecriptplasmid which contained a Nopaline Synthetase Promoter (PNOS) and theNopaline Synthetase Terminator (TNOS) fused to a transcriptionblock(TB). A PCR segment containing the entire PNOS-FIs:Trz:AHK4-TNOS-TBsequence with HindIII sites at the 5′ and 3′ ends was generated usingthe primers 5′-CTTCAAGCTTGATTCCCCGGATCATGAG-3′ (SEQ ID NO:27) and5′-CTTCAAGCTTAGAGAAATGTTCTGGCAC-3′ (SEQ ID NO:28). ThePNOS-FIs:Trz:AHK4-TNOS-TB was ligated into the HindIII site of thepCB302-3 vector containing ssTNT. The construct containingssTNT-FIs:Trg:PhoR in pCB302-3 was created using primers and techniquesessentially as above with the exception that a PhoR specific lowerprimer was used in making the FIs:Trg:PhoR fusion. The PhoR lower primersequence used is 5′-GCAAGCGATCGCTTAATCGCTGTTTTTGGCAA-3′ (SEQ ID NO:29).

TNT Induction of Arabidopsis Plants. TNT (ChemService, West Chester,Pa.) stock solution was prepared by dissolving TNT powder in DMSO. Finalsolutions containing different concentrations of TNT were prepared inwater. Plants were incubated in water (control) or water plus 10 uM TNTfor 16 hours.

GUS fluorometric assays. After incubation of plants with inducer of thegene circuit (CK, TNT, etc.), plants/leaves were ground in extractionbuffer (50 mM NaHPO₄, pH 7.0, 10 uM β-mercaptoethanol, 10 mM Na₂EDTA, pH8.0, 0.1% Sarcosyl, 0.1% Triton X-100). GUS Reaction: Protein extractcombined with extraction buffer was incubated and then stopped withreaction stop buffer (0.2 M Na₂CO₃) and 4-MU fluorescence read on a DyNAQuant 200 Fluorometer (Hoefer, Inc., San Francisco, Calif.) (Gallagher,S. R., 1992).

Plant material, transgenic plant production and growth conditions. Wildtype Arabidopsis thaliana (ecotype Columbia) were used for analysis andtransgenic plant production. Plasmids were assembled as described aboveand transferred into Agrobacterium tumefaciens strain GV3101 byelectroporation. Arabidopsis plants were transformed with thisAgrobacterium strain containing the assembled plasmids by floral dipmethod (Clough and Bent, 1998). Transgenic To plants were selected onstandard germination medium (GM) containing full strength Murashige andSkoog Salts (Murashige and Skoog, 1062). Leaves from wild-type tobaccoplants (cultivar SR1) were transformed using Agrobacterium tumefaciensstrain GV3101 by electroporation (same as the Arabidopsis plants).Leaves were co-cultivated with the Agrobacterium for 2-3 days.Transgenic tobacco plants were regenerated on standard MS selectionmedia supplemented with 50 mg/l Kanamycin (sigma) and 5 mg/l glufosinate(Crescent Chemical Co). Once shoots formed plants were transferred to MSmedia for rooting. Media is as above except that it lacked BAP and NAA(Dandekar et. al., 2005).

Induction of plant degreening and regreening. For the induction ofdegreening, 1 4-day old transgenic plants containing a single copy ofthe specific gene circuit were grown aseptically on MS medium with 50mg/L kanamycin but without sucrose. Individual plants were incubated in24-well Cellstar culture plates (Greiner Bio-one, Longwood, Fla.), eachwell containing 2 mL of liquid MS media without sucrose, supplemented(induced) or not (control) with the inducer, 10 μM 4-OHT. Induction ofthe degreening circuit typically started 1 hour into the 16 hour lightperiod, but results were the same regardless of when the inductionstarted. Plants were returned to the growth conditions described aboveand incubation continued for the different time periods or conditionsdescribed in the text. After degreening, plants were induced to regreenby incubation in 1 μM t-zeatin for 6 hours, then transferred to platescontaining MS media, and allowed to regreen for up to 7 days. Plantsregreened without use of the cytokinin, however, cytokinin treatmentenhanced the process.

Semi-quantitative RT-PCR analysis. Total RNA was isolated from wholeplants using the AURUM Total RNA Mini kit (Bio-Rad Laboratories,Hercules, Calif.), according to the instructions from the manufacturer.cDNA synthesis and PCR amplification were performed with 200 ng totalRNA, using the ACCESSQUICK RT-PCR System (Promega, Madison, Wis.) andgene-specific primers. Even though the RT-PCR system uses a DNase step,primers were designed to span an intron-exon junction except for GUN4,which lacks introns. The following primers were used: Cyclophilins,5′-GCGTTCCCTAAGGTATACTTCGAC-3′ (SEQ ID NO:30) and5′-CCCATGAGAACACACACCAAAC-3′ (SEQ ID NO:31);GUN4,5′-ACGCAAAATCTGGTTAAAAGTGAA-3′ (SEQ ID NO:32) and5′-TTGTGAGCGGTAAGTGTCCTAAAG-3′ (SEQ ID NO:33); POR,5′-TTGACCATCAAGGAACAGAGAA-3′ (SEQ ID NO:34) and5′-TATTTGTGTTTCCTGTTATAGA-3′ (SEQ ID NO:35); CHLASE,5′-TAGCCCCACAGTTGTGCAAATT-3′ (SEQ ID NO:36) and5′-AAGTCCGTTGGTGCGCATGGTG-3′ (SEQ ID NO:37); RCCR,5′-AATCTTCTCCGATTGATTTTGT-3′ (SEQ ID NO:38) and5′-CTAGAGAACACCGAAAGCTTCT-3′ (SEQ ID NO:39); PAO,5′-TCTATGAACAAAATTGAGTTAG-3′ (SEQ ID NO:40) and5′-CTACTCGATTTCAGMTGTACA-3′ (SEQ ID NO:41). PCR amplification consistedof 1 cycle at 95° C. for 2 min, followed by 25 cycles each of 95° C. for40 sec, 52° C. for 30 sec, 72° C. for 1 min, and a final extension stepat 72° C. for 5 min. Amplification products were separated on 2% agarosegels, and photographed under UV light using a Scion Image CaptureSystem.

Total chlorophyll and protein measurements. Chlorophyll was extracted in2.5 mM sodium phosphate buffered (pH 7.8) 80% acetone. Absorbance of theresulting solution was measured at 646.6 and 663.6 nm on a ShimadzuUV-1201 spectrophotometer, and total chlorophyll content (μg/mL) wascalculated using the formula: 17.76 A_(646.6)+7.34 A_(663.6) (Porra etal., 1989). Total proteins were estimated using the Bradford reagent(Bio-Rad, Hercules, Calif.) using BSA as a standard (Bradford, 1976).

Western blot analysis. Plants (3-5 per time point) were ground in liquidnitrogen and resuspended in 250 μL 12.5 mM sodium phosphate buffer (pH7.8). Protein concentration was determined using the Bradford reagent(Bradford, 1976). Protein samples were loaded based on equal freshweight on 12% SDS-PAGE gels, electrophoresed for 40 minutes at 200 V,and transferred to Hybond-P (Amersham Biosciences) membranes. Westernhybridizations were performed with the ECL plus western blottingdetection system (Amersham Biosciences). The GUN4 antibody was providedby Dr. R. M. Larkin (Michigan State University, East Lansing, Mich.) andused as recommended. Briefly, this consisted of blocking with 5%Blotting Grade Blocker non-fat dry milk (Bio-Rad) in PBS (pH 7.5),followed by incubation with the GUN4 antibody at a 1:1600 dilution andthe secondary antibody (HRP-conjugated anti-rabbit IgG, PierceBiotechnology, Rockford, Ill.) at a 1:20,000 dilution. The POR antibodywas provided by Dr. G. A. Armstrong (Ohio State University, Columbus,Ohio). Blocking conditions were similar to those described for GUN4,followed by incubation with POR antibody at 1:500 dilution and secondaryantibody at 1:10,000 dilution. Western blots were scanned and quantifiedusing a Molecular Dynamics Storm 840 system.

Microarray. 14 day-old transgenic plants bearing degreening circuit #3were harvested before induction, 24h and 48h post-induction with 10 μM4-OHT, and also after being allowed to regreen for 3 days. Treatmentswere replicated in two biologically independent experiments. Total RNAwas isolated from each sample (8 plants/treatment) using Aurum™ TotalRNA Mini kit (Bio-Rad Laboratories, Hercules, Calif.). Paired 2.5 pgaliquots from each treated and control RNA sample were reciprocallylabeled with either Cy3 or Cy5 dendrimers using the Array 900™ system(Genisphere, Hatfield, Pa.), such that RNA from each treatment waslabeled and hybridized (in parallel with a contrastingly labeled controlsample) four separate times, for a total of twelve microarrays balancedwith respect to treatment, dye, and biological replication. Microarrayswere spotted at high-density on SUPERAMINE slides (Telechem, Sunnyvale,Calif.) using amine-modified 70-mer oligonucleotide probes (ATH1 version1, Operon, Huntsville, Ala.) representing essentially every predictedgene in the Arabidopsis genome. Microarray signal intensities werequantified and analyzed using the TM4 software suite (Saeed et al.,2003). Raw signal intensities were normalized within array blocks usingthe Lowess function, and normalized, log₂ transformed signal intensitiesof >10 units (on a scale of 0-26) were subjected to statistical tests(t-test, one way ANOVA) to identify expression ratios that differedsignificantly from the mean.

Chlorophyll fluorescence imaging. To visualize early changes inphotosynthetic efficiency due to the degreening process, images ofchlorophyll fluorescence were obtained using the Fluor Cam (PhotonSystems Instruments, Brno, Czech Republic). Plants were induced todegreen as described above, and were compared to non-induced, as well aswild-type Columbia plants as a control. Plants were dark-adapted for 30minutes prior to fluorescence measurements. Data were obtained using thedefault fluorescence Quenching Analysis protocol, with the followingmodifications: a 30 sec dark pause after the F_(m) measurement was used;pulse fluorescences were subtracted for F_(m) measurement of the “dark”level and for F_(m) measurements during Kautsky induction. Data analysiswas performed using the manufacturer's software (Fluorcam v. 5.0). Timecourse plots of the relevant parameters were generated using MicrosoftExcel.

Reactive oxygen species (ROS) detection. To investigate the productionof ROS during the degreening process, the probe CM-H₂DCFDA (MolecularProbes, Eugene, Oreg.) was used. DCFDA is a cell-permeant indicatorwhich is nonfluorescent until the acetate groups are removed byintracellular esterases and oxidation occurs within the cell. ROSproduction was visualized under an Olympus FVX-IHRT Fluoview confocallaser scanning microscope using an Argon (488 nm) laser. Chlorophyllauto-fluorescence was visualized following excitation with a HeNe (543nm) laser.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

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1. A DNA construct comprising a plant operable promoter operably linkedto a nucleic acid sequence encoding a sensor protein, said proteincomprising a secretory sequence for directing the protein to theextracellular space of a plant cell and a binding region specific for atarget substance of interest, wherein said protein undergoes aconformational change when the target substance is bound.
 2. The DNAconstruct of claim 1, wherein the target substance is a nerve gas, aheavy metal, a poison, a pollutant, a toxin, an herbicide, a polycyclicaromatic hydrocarbon, a benzene, a toluene, a xylene, a halogenated(chloro, fluoro, and chlorofluoro) hydrocarbon, a steroid or otherhormone, an explosive, or a degradation product of one of the foregoingcompounds.
 3. The DNA construct of claim 2, wherein the encoded sensorprotein specifically binds trinitrotoluene.
 4. The DNA construct ofclaim 3, comprising the sequence of SEQ ID NO:8.
 5. A DNA constructcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a protein which comprises the following domains: aplasma membrane targeting signal sequence, an extracellular domain forbinding a sensor protein, a transmembrane domain and a histidine kinasedomain for phosphorylating a protein with nuclear shuttling ortranscriptional activating functions, wherein the histidine kinase isactivated when a sensor protein binds to the extracellular domain. 6.The DNA construct of claim 5, wherein the extracellular domain, thetransmembrane domain and the histidine kinase domain are derived fromone or more bacterial genes and, the membrane targeting signal sequenceis derived from a plant gene.
 7. The DNA construct of claim 1, whereinthe secretory sequence is from pollen extension-like protein (PEX). 8.The DNA construct of claim 6, wherein the membrane targeting signalsequence is from FLS2.
 9. The DNA construct of claim 5, wherein saidhistidine kinase domain comprises segments derived from a bacterium anda plant.
 10. The DNA construct of claim 9, wherein said histidine kinasedomain comprises segments from a bacterial histidine kinase and a plantArabidopsis histidine kinase (AHK4) protein.
 11. The DNA construct ofclaim 6, wherein the sequence encoding the histidine kinase is that ofSEQ ID NO:9.
 12. A DNA construct comprising a plant operable promoteroperably linked to a nucleic acid sequence encoding a detectable markeror a response gene, wherein the promoter is responsive to an internalsignal caused by an external target substance of interest, and whereinsaid detectable marker is expressed when an external target substance ofinterest is bound to a sensor protein.
 13. The DNA construct of claim12, wherein the detectable marker is a chlorophyll degradation enzyme ora functional fragment thereof.
 14. The DNA construct of claim 13,wherein the chlorophyll degradation enzyme is selected from the groupconsisting of red chlorophyll catabolite reductase (RCCR), pheophorbidea oxygenase (PaO), or chlorophyllase.
 15. The DNA construct of claim 13,further comprising a plant operable promoter responsive to an externaltarget substance of interest operably linked to a nucleic acid sequenceencoding an interfering RNA molecule specific for a chlorophyllbiosynthesis coding sequence.
 16. The DNA construct of claim 15, whereinthe chlorophyll biosynthesis coding sequence encodes chlorophyllsynthetase, protochlorophyllide oxidoreductase (POR) or genomeuncoupling 4 (GUN4), a gene regulating chlorophyll biosynthesis.
 17. TheDNA construct of claim 13, wherein the plant operable promoter comprisesa PhoB binding sequence.
 18. The DNA construct of claim 17, wherein theplant operable promoter is set forth in SEQ ID NO:1.
 19. A DNA constructcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a plant operable transcriptional activator, whereinthe transcriptional activator is activated when phosphorylated by ahistidine kinase.
 20. The DNA construct of claim 19, wherein the plantoperable transcriptional activator is that of SEQ ID NO:4.
 21. The DNAconstruct of claim 19, wherein the plant operable transcriptionalactivator is that of SEQ ID NO:11.
 22. A transgenic plant comprising: a.a first DNA construct comprising a plant operable promoter operablylinked to a nucleic acid sequence encoding a sensor protein, saidprotein comprising a secretory sequence for directing the protein to theextracellular space of a plant cell and a binding region specific for atarget substance of interest, wherein said protein undergoes aconformational change when the target substance is bound, and b. asecond DNA construct comprising a plant operable promoter operablylinked to a nucleic acid sequence encoding a protein which comprises thefollowing domains: a plasma membrane targeting signal sequence, anextracellular domain for binding a sensor protein, a transmembranedomain and a histidine kinase domain for phosphorylating a protein withnuclear shuttling or transcriptional activating functions, wherein thehistidine kinase is activated when a sensor protein binds to theextracellular domain, and c. a third DNA construct comprising a plantoperable promoter operably linked to a nucleic acid sequence encoding adetectable marker or a response gene, wherein the promoter is responsiveto the transcriptional activator protein, and wherein the detectablemarker is expressed-when an external target substance of interest isbound to a sensor protein.
 23. The transgenic plant of claim 22, whereinthe target substance of the DNA construct is a nerve gas, a heavy metal,a poison, a pollutant, a toxin, an herbicide, a polycyclic aromatichydrocarbon, a benzene, a toluene, a xylene, a halogenated (chloro,fluoro, and chlorofluoro) hydrocarbon, a steroid or other hormone, anexplosive, or a degradation product of one of the foregoing compounds.24. The transgenic plant of claim 22, wherein the encoded sensor proteinof the DNA construct specifically binds trinitrotoluene.
 25. Thetransgenic plant of claim 22, wherein the DNA construct comprises thesequence of SEQ ID NO:8.
 26. The transgenic plant of claim 22, whereinthe extracellular domain, the transmembrane domain and the histidinekinase domain of the DNA construct are derived from one or morebacterial genes, and the membrane targeting signal sequence is derivedfrom a plant gene.
 27. The transgenic plant of claim 22, wherein thesecretory sequence is from PEX.
 28. The transgenic plant of claim 22,wherein the membrane targeting signal sequence is from FLS2.
 29. Thetransgenic plant of claim 22, wherein the histidine kinase domain of theDNA construct comprises segments derived from a non-plant organism. 30.The transgenic plant of claim 22, wherein the histidine kinase domain ofthe DNA construct comprises segments derived from a non-plant organismand a plant
 31. The transgenic plant of claim 22, wherein the sequenceencoding the histidine kinase of the DNA construct is that of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:10 or SEQ ID NO:12.
 32. The transgenicplant of claim 22, wherein the detectable marker of the DNA construct isa chlorophyll degradation enzyme or a functional fragment thereof. 33.The transgenic plant of claim 22, wherein the plant loses detectablegreen color when the detectable marker is expressed.
 34. The transgenicplant of claim 32, wherein the chlorophyll degradation enzyme of the DNAconstruct is selected from the group consisting of red chlorophyllcatabolite reductase (RCCR), pheophorbide a oxygenase (PaO), orchlorophyllase.
 35. The transgenic plant of claim 22, further comprisinga plant operable promoter responsive to a transcription activatorprotein operably linked to a nucleic acid sequence encoding aninterfering RNA molecule specific for a chlorophyll biosynthesis codingsequence.
 36. The transgenic plant of claim 35, wherein the chlorophyllbiosynthesis coding sequence encodes chlorophyll synthetase,protochlorophyllide oxidoreductase (POR) or GUN4, a gene regulatingchlorophyll biosynthesis.
 37. The transgenic plant of claim 22, whereinthe plant operable promoter of the DNA construct comprises a PhoBbinding sequence.
 38. The transgenic plant of claim 22, wherein theplant operable promoter of the DNA construct is set forth in SEQ IDNO:1.
 39. The transgenic plant of claim 22 which further comprises afourth DNA construct comprising a nucleic acid encoding a chlorophylldegradation enzyme or a functional fragment thereof operably linked to aplant operable promoter responsive to the transcription activatorprotein, and wherein said promoter is not in nature associated with saidsequence encoding a chlorophyll degradation enzyme.
 40. The transgenicplant of claim 22 which further comprises a fourth DNA constructcomprising a plant operable promoter operably linked to a nucleic acidsequence encoding a plant operable transcriptional activator, whereinthe transcriptional activator is activated when phosphorylated by ahistidine kinase.
 41. The transgenic plant of claim 22, wherein thedetectable marker is a functional RNA.
 42. The transgenic plant of claim41, wherein the functional RNA is an interfering RNA molecule.
 43. Thetransgenic plant of claim 42, wherein the functional RNA inhibitsexpression of a chlorophyll biosynthesis coding sequence.
 44. Thetransgenic plant of claim 43, wherein the chlorophyll biosynthesiscoding sequence encodes chlorophyll synthetase, protochlorophyllideoxidoreductase (POR) or GUN4, a gene regulating chlorophyllbiosynthesis.
 45. The transgenic plant of claim 22, wherein thedetectable marker is a chlorophyll degradation enzyme.
 46. Thetransgenic plant of claim 45, wherein the chlorophyll degradation enzymeis red chlorophyll catabolite reductase (RCCR), pheophorbide a oxygenase(PaO), or chlorophyllase.
 47. The transgenic plant of claim 41, whereinthe detectable marker is a α-glucuronidase, a β-galactosidase or a greenor yellow fluorescent protein.
 48. The transgenic plant of claim 22,wherein the transcription activator protein comprises a responseregulator domain.
 49. The transgenic plant of claim 48, wherein theresponse regulator domain is derived from PhoB.
 50. The transgenic plantof claim 22, wherein the transcription activator protein is a PhoB:VP64translational fusion protein.
 51. The transgenic plant of claim 50,wherein the sequence encoding the PhoB:VP64 protein is given in SEQ IDNO: 4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:11.
 52. The transgenicplant of claim 41, wherein the detectable marker is a functional RNAwhich inhibits expression of a chlorophyll biosynthesis coding sequence.53. The transgenic plant of claim 52, wherein the plant loses greencolor due to inhibition of chlorophyll biosynthesis and enhancedbreakdown of chlorophyll upon induction of a gene encoding a chlorophylldegradation enzyme.
 54. The transgenic plant of claim 52, wherein theenhanced breakdown of chlorophyll is achieved by expressing at least oneenzyme selected from the group consisting of red chlorophyll catabolitereductase (RCCR), pheide a oxygenase (PaO), and chlorophyllase.
 55. Thetransgenic plant of claim 52, wherein the inhibition of chlorophyllbiosynthesis is achieved by inhibiting expression of at least one enzymeselected from the group consisting of protochlorophyllide oxidoreductase(POR), chlorophyll synthetase and GUN4.
 56. The transgenic plant ofclaim 53, wherein the inhibition of POR is achieved by producing aninterfering RNA molecule that contains a sequence derived from thecoding sequence of POR.
 57. The transgenic plant of claim 52, whereinthe plant loses green color by inhibiting POR and stimulating RCCR andchlorophyllase.
 58. A method for detecting an external substance ofinterest, said method comprising the step of exposing the transgenicplant of claim 22 to an external substance of interest and detecting achange resulting from expression of the detectable marker.
 59. Themethod of claim 58, wherein the detectable marker is a functional RNA.60. The method of claim 59, wherein the functional RNA is an interferingRNA molecule.
 61. The method of claim 60, wherein the functional RNAinhibits expression of a chlorophyll biosynthesis coding sequence. 62.The method of claim 61, wherein the chlorophyll biosynthesis codingsequence encodes chlorophyll synthetase, protochlorophyllideoxidoreductase (POR) or a GUN4, a gene regulating chlorophyllsynthetase.
 63. The method of claim 58, wherein the detectable marker isa chlorophyll degradation enzyme.
 64. The method of claim 63, whereinthe chlorophyll degradation enzyme is red chlorophyll catabolitereductase (RCCR), pheophorbide a oxygenase (PaO), or chlorophyllase. 65.The method of claim 61, wherein the detectable marker is aβ-glucuronidase, a β-galactosidase or a green or yellow fluorescentprotein.
 66. The method of claim 58, wherein the transcription activatorprotein is a PhoB protein or is derived from a PhoB protein.
 67. Themethod of claim 66, wherein the transcription activator protein is aPhoB:VP64 translational fusion protein.
 68. The method of claim 66,wherein the sequence encoding the PhoB:VP64 protein is given in SEQ IDNO: 4 or SEQ ID NO:5.
 69. The method of claim 58, wherein the change isdegreening of the transgenic plant.
 70. The method of claim 69, whereinthe degreening of the transgenic plant is detected visually or bydetecting properties selected from the group consisting of chlorophyllfluorescence, photosynthetic properties and properties related toreactive oxygen species and their damage.
 71. The method of claim 70,wherein said degreening is detected by imaging selected from the groupconsisting of hyper-spectral imaging, infra-red imaging, near-infra-redimaging and multi-spectral imaging.
 72. The method of claim 69, whereinthe transgenic plant regreens after removal of the external substance ofinterest.